Effects of ACE2 and Co-factors on the Mechanism of SARS-CoV-2 Infection Among Colorectal Cancer Patients

Background: The infection of host cells by SARS-CoV-2 is mediated by ACE2 and its co-factors, including TMPRSS2 and FURIN. Patients with cancer are highly susceptible to COVID-19 and exhibit a higher risk of severe progression. However, the mechanism of SARS-CoV-2 infection on colorectal cancer prognosis is largely unknown. Here, we investigated the role of key genes related to SARS-CoV-2 in colorectal cancer. Methods: RNA sequencing (RNA-Seq), proteomics and single-cell RNA-Seq were used to pro�le the expression of ACE2, TMPRSS2, and FURIN in colorectal cancer. A pseudovirus was used to transfect colon cells to compare the infection rate of SARS-CoV-2 between different cell types. The TIMER database was used to analyze the association of candidate genes with immune in�ltration in colorectal cancer. Cox regression model was performed to evaluate genetic effects of ACE2 on colorectal cancer prognosis. Results: ACE2 was upregulated in colorectal cancer tissues, while TMPRSS2 and FURIN were downregulated. Increased ACE2 expression was signi�cantly associated with a decreased tumor mutational burden. Furthermore, ACE2 promoted the SARS-CoV-2 infection in colon cancer cells than that in normal colon epithelial cells, as well as that ACE2 could affected the immune in�ltration level and prognosis in colorectal cancer patients. Moreover, individuals with genetic variants in ACE2 exhibited poor overall survival of colorectal cancer. Conclusions: These results are the �rst time to identify that ACE2 and its co-factors are associated with the infection and survival of colorectal cancer patients with SARS-CoV-2. Our �ndings would be helpful for extending the mechanisms in SARS-CoV-2 infection among colorectal cancer.


Background
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus leading to coronavirus disease (COVID- 19), is a new major disease threat to humans.The rapid spread of  in approximately 200 countries and territories has created a global epidemic affecting more than 200 million patients in most countries of the world, with more than four million con rmed deaths.
Recent studies have reported that patients with cancer show a higher risk of infection with SARS-CoV-2, more severe adverse events and a poorer prognosis than those without cancer [1,2].Notably, cancer history is the only independent risk factor for COVID-19 among common comorbidities [3].This is due to the systemic immunosuppressive state caused by several anticancer treatments [4,5].Lymphopenia or neutropenia in cancer patients could also cause worse outcomes of 7].Colorectal cancer is one of the most common cancers and is the second most common cause of cancer-related mortality [8].As reported recently, the levels of IL-6, numbers of neutrophils and ratio of neutrophils to lymphocytes (NLR) in the mild group are signi cantly lower than those in the severe group of colorectal cancer patients with COVID-19 [9].In addition, SARS-CoV-2 can infect intestinal organoids according to the identi cation of live virus in stool samples from COVID-19 patients [10,11].However, there is still a large gap in the understanding of biological mechanisms of colorectal cancer in association with SARS-COV-2.SARS-CoV-2 was previously shown to enter host cells by binding to angiotensin-converting enzyme 2 (ACE2) [12,13].ACE2 can cleave both angiotensin 1 and 2, which is the only experimentally con rmed SARS-CoV-2 receptor [14,15].Additionally, transmembrane protease serine 2 (TMPRSS2) and FURIN (FURIN), which are two co-factors of ACE2, can facilitate the cleavage of the SARS-CoV-2 spike glycoprotein [12,16].Although ACE2 and its co-factors have been reported to be differentially expressed in various tissues to regulate the susceptibility of cancer patients to SARS-CoV-2 infection [17,18], the expression and functional role of ACE2 and its co-factors in colorectal cancer remain to be explored.
In this study, we explored the expression pattern of ACE2 and its co-factors at mRNA and protein level in colorectal cancer, as well as their effects on infection rate of SARS-CoV-2 between colorectal cancer and normal colon epithelial cells.Furthermore, the regulation of immune in ammation and the genetic effects of ACE2 and its co-factors were analyzed to identify the mechanisms of host factors in the induction of SARS-CoV-2 infection and ultimately provide treatment strategies for SARS-CoV-2 infections in colorectal cancer patients.

Study populations and samples
All the participants in this study were unrelated Han Chinese individuals and provided written informed consent.The detailed information of this study population is described in the Supplementary Materials and Methods.In addition, a total of 544 patients were recruited for a follow-up investigation performed via telephone every six months.The characteristics of the subjects are shown in Table S1.This study was approved by the institutional review board of Nanjing Medical University, China.

RNA isolation and RNA sequencing (RNA-Seq)
Total RNA was extracted using TRIzol Reagent (Invitrogen, CA, USA) according to the manufacturer's protocol.The concentration and integrity of RNA were con rmed using a Nanodrop 2000 spectrometer (ThermoFisher) and an Agilent 2100 Bioanalyzer (Agilent, CA, USA).The detailed protocol for RNA-Seq is described in the Supplementary Materials and Methods.

Protein extraction and tryptic digestion for tandem mass tag (TMT) analysis
The collected tissue samples were xed in RNAlater stabilization solution (Invitrogen, Carlsbad, CA) and stored in a -80°C refrigerator for protein extraction.The detailed protocols for protein extraction and the TMT experiment are described in the Supplementary Materials and Methods.

Gene expression analysis in different tissues
mRNA expression pro les were downloaded from datasets of The Cancer Genome Atlas (TCGA) (http://cancergenome.nih.gov/)[19], Gene Expression Omnibus (GEO) and Gene Expression Pro ling Interactive Analysis (GEPIA) [20].Immunohistochemical images and protein expression pro les were obtained from the Human Protein Atlas (HPA) database [21].The single-cell RNA-Seq data for colorectal cancer tissues were obtained from GSE81861 [22].The details of the data sources and the processing methods for the gene expression analysis are provided in the Supplementary Materials and Methods.

Cell transfection and SARS-CoV-2 pseudovirus treatment experiments
The details of the cell lines and cell cultures are described in the Supplementary Materials and Methods.For the stable overexpression of ACE2, cells were transfected with the GV358 lentivirus vector or a negative control (GeneChem, China) expressing green uorescent protein (GFP) as a marker.The stable effect of ACE2 overexpression was con rmed by the observation of GFP under a uorescence microscope and Western blotting.The SARS-CoV-2 pseudovirus was produced with the pLVX-EGFP-Luciferase reporter.To evaluate the infection ability and rate of SARS-CoV-2 in different types of colon cells, FHC, HCoEpiC, SW620 and LoVo cells were used to conduct luciferase activity assays and immuno uorescence staining after the stable overexpression of ACE2.The protocols for these experimental procedures are available in the Supplementary Materials and Methods.
Tumor Immune Estimation Resource (TIMER) analysis TIMER is a web server for the systematic analysis of immune in ltrates in different cancers [23,24].This comprehensive resource provides six major analytic modules for exploring the associations between immune in ltrates and different factors, such as gene expression, somatic mutation and clinical outcomes.We analyzed the association of the expression of selected genes with the abundance of in ltrating immune cell types.Expression scatter plots between selected genes were generated using the correlation module, together with Spearman's correlation and the estimated statistical signi cance.

Genetic variants and somatic mutation analysis
The details of the SNP genotyping methods and the somatic mutation analyses are described in the Supplementary Materials and Methods.

Statistical analysis
All continuous data are presented as the mean ± SD.A two-sided P value of less than 0.05 was regarded as statistically signi cant.The more details of statistical analysis are described in the Supplementary Materials and Methods.

Expression pattern of ACE2, TMPRSS2 and FURIN across different colorectal tissues
We rst assessed the mRNA expression pattern of ACE2 in colorectal cancer tumors using in-house RNA-Seq, and found that the mRNA expression level of ACE2 was signi cantly higher in tumor tissues than in adjacent normal tissues (Figure 1A).However, the distinct expression was not validated in the TCGA database (Figure S1A); and even could not be found in meta-analysis of ACE2 expression using the combined in-house RNA-Seq dataset, TCGA and GEO datasets (Figure S1B).Therefore, we used TMTbased global proteomics to evaluate the protein expression of ACE2 in paired colorectal clinical tissues.
The protein expression of ACE2 was higher in colorectal tumor tissues than in paired normal tissues (Figure 1B), which was also supported by immunohistochemical images that high staining and strong intensity of ACE2 existed in colon and rectum tumor tissues (Figure 1C).Subsequently, we analyzed the mRNA expression of TMPRSS2 and FURIN between colorectal cancer tissues and normal tissues via a meta-analysis of the RNA-Seq results and TCGA and GEO datasets.As shown in Figure 1D-E, the expression of both TMPRSS2 and FURIN was signi cantly decreased in colorectal cancer tissues compared with normal tissues.The immunohistochemical images of TMPRSS2 and FURIN staining also revealed that TMPRSS2 and FURIN were downregulated in colon tumor tissues (Figure 1F-G).Additionally, the heat map illustrated that these two genes were co-lower expressed in colorectal cancer tissues compared to paired normal tissues (Figure 1H).Furthermore, the expression levels of TMPRSS2 and FURIN gradually decreased along with malignant progression in the course of normal, adenoma and tumor (Figure 1I-L).
Given that ACE2 cooperating with co-factors TMPRRS2 and FURIN, plays key roles in regulating the binding and entry a nity of SARS-CoV-2, we further conducted Spearman's correction analysis between ACE2 and its co-factors.As shown in Figure S1C-D, the expression of ACE2 was positively correlated with TMPRRS2 and negatively corrected with FURIN.A positive correlation was also detected between the expression levels of the two co-factors (Figure S1E).Therefore, we hypothesized that three genes were coexpressed and interacted in colorectal tumor tissues.
We then examined the role of onset age in the expression of these three genes.Higher ACE2 expression and lower FURIN expression were signi cantly related to late-onset colorectal cancer, while no association was found between onset age and TMPRSS2 expression (Figure S2).We also found no signi cant differences in the tumor site or tumor stage related to the expression of these three genes (Figure S2).

RNA and protein expression of ACE2 and its co-factors in pan-cancer tissues
Based on the GEPIA2 and HPA databases, we compared the mRNA and protein expression of ACE2 and its co-factors in different tissues.Notably, FURIN mRNA showed high abundance in most types of tissues compared with that of ACE2 and TMPRSS2 (Figure S3).However, the protein immunoreactivity of TMPRSS2 in most types of tumor tissues was generally weaker or negative compared with that of ACE2 and FURIN (Figure S4).ACE2 protein expression was observed to show moderate to strong cytoplasmic and membranous immunoreactivity in colorectal and renal cancers.

Co-expression pattern of ACE2 and its co-factors in colorectal cancer cells determined by single-cell RNA-Seq analysis
We next carried out single-cell RNA-Seq to evaluate the expression pattern of ACE2 and its co-factors in colorectal cancer tissues.As shown in Figure 2A-D, among colorectal tumor cell clusters, the expression of ACE2 and TMPRSS2 was heterogeneous, with some cells co-expressing both ACE2 and TMPRSS2 (9.33%), while some tumor cell clusters expressed only ACE2 (3.2%) or only TMPRSS2 (31.2%).Similarly, 15.2% of colorectal tumor cells co-expressed both ACE2 and FURIN, while 9.87% and 2.67% of cell clusters expressed only ACE2 or only TMPRSS, respectively (Figure 2E-H).These results provided the coexpression pattern of ACE2 and the co-factors in the colorectal tumor cells.

The infection rate of SARS-CoV-2 in colon cells
To evaluate the infection rate of SARS-CoV-2 in colon cell lines, we rst tested the ACE2 expression level of normal colon epithelial cells (FHC and HCoEpiC) and colon tumor cells (SW620 and LoVo).None of these cells showed ACE2 expression that was detectable by Western blotting.Therefore, cell lines with stable overexpression of ACE2 were cultured, and the transduction e ciency was con rmed by GFP uorescence and Western blotting (Figure 3A-B).Subsequently, we transfected the SARS-CoV-2 pseudovirus into cells and performed luciferase reporter assays.We found that relative luciferase activity was signi cantly increased in colorectal tumor cells compared with normal colon epithelial cells (Figure 3C).Finally, higher positive staining of SARS-CoV-2 was detected in colorectal tumor cells by immuno uorescent staining, while lower positive staining was observed in normal colon epithelial cells (Figure 3D).For the rst time, we identi ed a higher infection rate of SARS-CoV-2 in ACE2-positive colorectal tumor cells than in normal colon epithelial cells.
Association of ACE2 and co-factor expression with immune in ltration in colorectal cancer tissues Given the association of crucial gene expression with immune in ltration levels in colorectal cancer, we investigated whether ACE2 and its co-factors were associated with immune in ltration in different types of immune cells by using the TIMER algorithm (Table S2).We observed a signi cant negative association of tumor purity with FURIN (r = -0.180,P = 2.58 × 10 -4 ), but no signi cant association of tumor purity with ACE2 or TMPRSS2 was found (Figure 4).Interestingly, we found that ACE2 expression presented a signi cant negative association with the in ltrating levels of neutrophils (r = -0.115,P = 2.11 × 10 -2 ) and dendritic cells (r = -0.097,P = 5.16 × 10 -2 ) in colon cancer tissues (Figure 4).Moreover, the expression of TMPRRS2 and FURIN presented a signi cant positive association with the in ltrating levels of neutrophils (r = 0.124, P = 1.29 × 10 -2 in TMPRSS2; r = 0.225, P = 5.12 × 10 -6 in FURIN) and dendritic cells (r = 0.121, P = 1.53 × 10 -2 in TMPRSS2; r = 0.288, P = 4.20 × 10 -9 in FURIN) in colon cancer tissues (Figure 4).These ndings revealed that ACE2 and its co-factors play speci c roles in immune in ltration in colorectal cancers, especially in of neutrophil and dendritic cells.

Prognostic analysis of ACE2 and its co-factors in colorectal cancer tissues
To explore the prognostic value of ACE2 and its co-factors in colorectal cancer, we performed Kaplan-Meier analysis based on the TCGA database.We found that low expression of ACE2 was signi cantly associated with a poor overall survival time (P = 0.028) (Figure 5A), while no signi cant association was observed between the overall survival time and the expression levels of TMPRSS2 (P = 0.073) or FURIN (P = 0.220) (Figure 5B-C).In addition, the overall survival time of patients in the higher-risk-score group was obviously longer than that of patients in the low risk score group for the combination of ACE2 and FURIN (P = 0.046) (Figure 5D).However, we only detected a weak association between a longer overall survival time and the higher risk score group for the combination of ACE2 and TMPRSS2 (P = 0.069) (Figure 5E) or the combination of three genes (P = 0.058) (Figure 5F).We also visualized the association between survival status and risk score rank as well as overall survival (Figure 5G-I).

Association of genetic variants in ACE2 and its co-factors with colorectal cancer risk and survival
Given that genetic variants in cancer-related genes are associated with colorectal cancer, we sought to investigate the genetic effect of SNPs in ACE2 and its co-factors on colorectal cancer susceptibility and prognosis.A summary of the experimental design and work ow is shown in Figure S5.A total of 19 candidate SNPs in ACE2 and its co-factors were included in the genotyping analysis after quality control and function prediction.Among these SNPs, no signi cant association was detected between candidate SNPs and the susceptibility of colorectal cancer (Table S3).
We then used 540 patients with follow-up information to con rm the genetic effect of candidate SNPs in predicting the prognosis of colorectal cancer (Table S4).Only two SNPs in the ACE2 gene were signi cantly associated with colorectal cancer survival time (HR = 1.40,P = 0.017 for rs2106809 and HR = 1.34,P = 0.038 for rs2285666) (Figure 6A-B).We also combined these two SNPs according to the number of risk alleles.As shown in Figure 6C, colorectal cancer patients with 1-4 risk alleles presented signi cantly poorer overall survival than those with 0 risk alleles (P < 0.05).Due to these two SNPs located in the ACE2 gene, we performed the functional annotation of rs2106809 and rs2285666 and the SNPs showing high linkage disequilibrium (rs4646142 and rs184697926) based on the Encyclopedia of DNA Elements (ENCODE) and Roadmap Epigenome Projects.The region encompassing these four SNPs was predicted in silico to present possible enhancer activity (Figure 6D), which indicated that genetic variants in the ACE2 gene could be useful in prognosis prediction for colorectal cancer.

Somatic mutation patterns of ACE2 and its co-factors in colorectal cancer tissues
We extracted mutational signatures from the TCGA database to identify somatic mutation patterns in ACE2 and its co-factors in colorectal cancer patients.As demonstrated in Figure S6A and Table S5, missense mutations in ACE2 (73.33%),FURIN (75.00%) and TMPRSS2 (83.33%) are particularly common in colorectal cancer tissues.Interestingly, we observed a signi cant increase in the mutation frequency of the ACE2 gene in the early-onset colorectal cancer group compared with the late-onset colorectal cancer group (P = 0.023) (Table S6).We also detected a decreasing trend in ACE2 expression in patients with ACE2 mutations (P = 0.059) (Figure S6B).
TMB is an emerging biomarker for the immunotherapy response of cancers.In this study, a higher TMB was detected in individuals with ACE2 mutations compared with those without ACE2 mutations (P = 3.32 × 10 -5 ) (Figure S6C).We also found a signi cant decrease in TMB associated with increased ACE2 expression (r = -0.149,P < 0.001) (Figure S6D).Collectively, these results highlight the important roles of somatic mutations in the ACE2 gene and TMB in colorectal cancer progression.

Discussion
The ongoing outbreak of COVID-19 has affected more than 200 countries.Patients with cancer are highly susceptible to COVID-19 and show a higher risk of severe adverse events and death [2,25].However, there is limited available information about the mechanisms underlying the outcomes of colorectal cancer patients who contract COVID-19.In this study, we found that ACE2 was upregulated, while TMPRSS2 and FURIN were downregulated in colorectal cancer tissues.In addition, the infection rate of SARS-CoV-2 was signi cantly higher in ACE2-positive colon tumor cells.The expression of three genes was associated with the immune in ltration level and the overall survival time of colorectal cancer patients.Genetic factors related to ACE2 were also found to play key roles in colorectal cancer prognosis.Our study ndings suggest that ACE2 and its co-factors participate in the mechanisms underlying the association of colorectal cancer with COVID-19 (Fig. 7).SARS-CoV-2, a single-stranded positive-sense RNA virus of the β-Coronaviridae family, has been con rmed to enter different mammalian cell types showing the expression of the ACE2 receptor [26,27].ACE2 is abundantly expressed in digestive tract organs [28,29].Recent research suggested that the expression of ACE2 gradually increases from the normal colon epithelium to adenoma and colorectal tumor tissues [30].We found that ACE2 was upregulated in colorectal cancer tissues and was related to the overall survival time of colorectal cancer patients.SARS-CoV-2 can interact with ACE2 to infect host cells via the S1 receptor-binding domain (RBD) of its surface spike glycoprotein [12,13].Previous studies have used authentic and pseudovirus SARS-CoV-2 neutralization assays to obtain antibodies that can signi cantly protect host cells from SARS-CoV-2 infection [31][32][33].Moreover, SARS-CoV-2 could bind platelet ACE2 to induce thrombosis in COVID-19 [34].We found that colon cancer cells are more likely to be infected by SARS-CoV-2 than normal colon epithelial cells.This provides novel evidence for understanding the susceptibility and prognosis of colorectal cancer patients after infection with COVID-19.
TMPRSS2 and FURIN are the major proteases that cleave the SARS-CoV-2 spike glycoprotein [12,35].A previous study showed that the entry of SARS-CoV-2 could be successfully blocked by blocking the protease activity of TMPRSS2 in colon cells [12], which indicated that SARS-CoV-2 requires other cofactors, including TMPRSS2 and FURIN.Moreover, the cleavage of the spike glycoprotein at the FURIN cut site could induce the binding ability of SARS-CoV-2 to ACE2 [36].Notably, TMPRSS2 and FURIN have special impacts on the cellular neighborhood, signi cantly increasing the susceptibility of ACE2-positive cell clusters to infection with SARS-CoV-2 [37].According to our data, TMPRSS2 and FURIN are downregulated in colorectal cancer tissues.In addition, the co-expression of these two genes with ACE2 may show potential for predicting the overall survival of colorectal cancer patients.However, the involvement of ACE2 and its co-factors in the infection pathway of SARS-CoV-2 remains to be investigated by future experimental methods.
Recent studies have suggested that severe adverse events in COVID-19 patients are dramatically related to an increase in innate immune cells such as neutrophils, indicating that immunopathology is involved [38,39].Previous in vivo experiments con rmed that ACE2 suppresses lung in ammation and neutrophil in ltration by reducing the activity IL-17 signaling [40].In addition, signi cant positive and negative correlations have been con rmed between ACE2 expression and IL-17(Th17)-dependent and Th2dependent epithelial gene signatures, respectively [41,42].Interestingly, our data demonstrated that ACE2 was signi cantly negatively correlated with the in ltrating levels of neutrophils and dendritic cells.These ndings may explain the association of abnormal immune in ltration levels with the outcomes of COVID-19-infected colorectal cancer patients.
Genetic variants in cancer-related genes are associated with the risk and prognosis of cancer.Numerous studies on genetic variants in ACE2 have focused on its association with cardiovascular disease and stroke [43,44], but there is a lack of reports on its relationship to colorectal cancer.Our study indicated that rs2106809 and rs2285666 in ACE2 were associated with the overall survival of colorectal cancer patients.Bioinformatics analysis demonstrated that rs2106809 could create an intronic exonic splicing enhancer (ESE) site to alter the splicing e ciency of ACE2 [45].In addition, the SNP rs2285666, which is located in a splice region of ACE2, might show strong functional regulation of ACE2.TMB, in concert with PD-L1 expression, is emerging as a potential biomarker.Patients with a high TMB have been observed to present an association with the response to PD-1/PD-L1 blockade, including melanoma and colorectal cancer [46,47].Chan et al. identi ed TMB development as an immunotherapy biomarker across several cancer types [48].In addition, TMB is associated with the prognosis and the response to immune checkpoint inhibitors in colorectal cancer patients [49,50].Our results further identi ed a signi cant decrease in TMB associated with increased ACE2 expression, which suggested that a higher TMB shows a signi cant correlation with colorectal cancer progression.

Conclusions
Taken together, this is the rst study to identify that higher expression of ACE2 and lower expression of its co-factors signi cantly increase the susceptibility of ACE2-positive colorectal cancer cells to SARS-CoV-2 infection.These genes are also associated with abnormal immune in ltration levels and the prognosis of colorectal cancer patients.Our ndings could be helpful for further exploring the mechanism underlying the association of colorectal cancer with COVID-19.

Ethics approval and consent to participate
This study was approved by the review board of Nanjing Medical University.

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