Genomic, epigenomic, and immune subtype analysis of CTSL/B and SARS-CoV-2 receptor ACE2 in pan-cancer

SARS-coronavirus 2 (SARS-CoV-2) has been spreading widely and posing an international challenge for both healthcare and society. At present, cancer has been identified as an individual risk factor for COVID-19. Angiotensin converting enzyme 2 (ACE2) and Cathepsin L/Cathepsin B (CTSL/B), which act as the receptor and entry-associated proteases of SARS-CoV-2 respectively, are pivotal for SARS-CoV-2 infection. To investigate the possible SARS-CoV-2 infection risk of pan-cancer, we analyzed the genetic alterations, RNA expression, DNA methylation, and the association with immune subtypes of ACE2 and CTSL/B with the prognosis in pan-cancer. Results showed the upregulation of CTSL/B and ACE2 in Pancreatic adenocarcinoma (PAAD) and Stomach adenocarcinoma (STAD) and demonstrated a positive correlation between copy number alteration (CNA) and gene expression for CTSB in PAAD and STAD. Hypomethylation and a negative correlation of gene expression and methylation for CTSB were detected in PAAD. In addition, ACE2 and CTSL/B are overexpressed in the IFN-gamma immune subtype of ovarian serous Cystadenocarcinoma (OV), Cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), and Bladder urothelial carcinoma (BLCA). Our study presents a bioinformatics assessment for the potential risk of SARS-CoV-2 infection in pan-cancer.

AGING viral membranes and viral activation for viral infection. SARS-CoV-2 uses the ACE2 receptor for entry [3]. The S1/S2 cleavage site of SARS-CoV-2 is between the threonine and methionine at positions 696 and 697, and is identical to that of SARS-CoV which has been shown to be cleaved by CTSL [4]. SARS-CoV takes advantage of the endosomal cysteine proteases CTSL and CTSB. In healthy individuals those organs including lung, heart, kidney, bladder and oesophagus which express a high level of ACE2 seem to be more vulnerable to SARS-CoV-2 infection [5]. Hence, the expression and distribution of ACE2 and CTSL/B may explain the different susceptibility to SARS-Cov-2 infection for pancancer. To address this possibility, we performed a comprehensive analysis of CTSL/B and ACE2 expression by using genomic, transcriptomic and epigenomic data of pan-cancer so as to provide a reference for the assessment of SARS-CoV-2 infection in pan-cancer.  Figure 1A, 1B). Further analysis revealed that ACE2 and CTSL/B are all highly expressed in PAAD and STAD ( Figure 1C, 1D). Oncomine analysis [7] verified the upregulation (p<0.05, fold change>1.5) of CTSL/B in PAAD and STAD when compared to normal tissues (Supplementary Tables 1, 2). With respect to ACE2 in the Oncomine analysis, the small sample size likely contributes to the lack of significant overexpression in PAAD and STAD as compared to normal tissues.
CNA, mutation and DNA methylation can prompt the variation of gene expression. CNA includes amplification, gain, diploidy, shallow deletion and deep deletion. c-BioPortal analysis [8] based on The Cancer Genome Atlas (TCGA) data indicates that genomic amplification increased the gene expression of CTSB in STAD and PAAD ( Figure 1E and Supplementary Figure  2, R>0.2, p<0.05). For CTSL in STAD, its expression could be elevated by genomic amplification as well ( Figure 1E). For ACE2, DNA mutation acts as the dominant factor in regulating gene expression in STAD and PAAD (Supplementary Figure 1B). However, the correlation analysis between gene expression and CNA/mutation showed neither ACE2 nor CTSL expression is relevant to mutation or CNA (Supplementary Figures 3, 4). For other types of tumors, the most frequent DNA alteration of the CTSL gene is DNA mutation in Uterine Corpus Endometrial Carcinoma (UCEC), ESCA, DLBC and Melanoma ( Figure 1E), and followed by amplification in Sarcoma (SARC), Adrenocortical carcinoma (ACC), UCEC and ESCA. For CTSB, deep deletion in Uterine Carcinosarcoma (UCS), Liver hepatocellular carcinoma (LIHC), BLCA and OV, is the most frequent DNA alteration, followed by amplification in ESCA, STAD, PAAD and DLBC. For ACE2, the most frequent DNA alteration is mutation in UCEC, UCS, STAD and MEL (Supplementary Figure 1B). To explore the influence of epigenetic modification on the mRNA levels for CTSL/B and ACE2 in PAAD and STAD, seven, fourteen and six CpG probes targeting the promoters of the CTSL, CTSB and ACE2 genes, respectively, using Methylation450k profiles were utilized to recapitulate the DNA methylation levels (Supplementary Table 3) in the DiseaseMeth database [9]; the results demonstrated that DNA methylation of CTSL/B is significantly decreased in PAAD but not in STAD when compared with normal tissues ( Figure 1F and Supplementary Figure 5). However, the analysis based on DNMIVD [10] showed that gene expression of CTSB rather than CTSL and ACE2 is negatively correlated with DNA methylation in PAAD and STAD (Supplementary Tables 4-6, R<-0.1, p<0.05). Together, the analyses suggest that both DNA methylation and CNA could influence CTSB gene expression in PAAD, while only CNA can affect CTSB gene expression in STAD. For proteomic analysis, we analyzed the expression variation of ACE2 and CTSL/CTSB with TCGA proteomics data, which includes measuring the expression of 261 proteins; unfortunately, these three proteins (ACE2, CTSL and CTSB) were not included in the 261 proteins.
We further studied the involvement of ACE2 and CTSL/B in cancer immune subtypes including C1 (wound healing), C2 (IFN-gamma dominant), C3 (inflammatory), C4 (lymphocyte depleted), C5 (immunologically quiet) and C6 (TGF-b dominant) in pan-cancer (Supplementary Figure 6) according to TISIDB [11]. As compared with other types of tumors, BRCA, HNSC, OV, KIRC, CESC and BLCA present greater differential ACE2 expression. Combining the immune subtype analysis of ACE2 and CTSL/B in the six tumor types, we found that ACE2 and CTSL/B are most highly overexpressed in the IFN-gamma dominant immune subtype of OV, CESC and BLCA ( CTSL/B have been reported to be a vital regulators in proliferation, metastasis [12], invasion [13] and prognosis [14] in various types of cancers. For instance, Cathepsins B and L could drive the invasive growth of human melanoma cells [15], and overexpression of CTSL is a marker of invasion and metastasis in ovarian cancer [16], together manifesting a potential capacity for the two proteases CTSL/B as malignant phenotype markers for pan-cancer. In addition, the finding that ACE2 overexpression presents a poor prognosis in LGG, consistent with previous reports [17].
Regarding COVID patients with malignancies, LUAD patients with overexpressed ACE2 were found to have a higher incidence of COVID-19 [18]. Herein, we showed that both ACE2 and CTSL/B are upregulated in PAAD and STAD and are overexpressed in IFN-gamma immune subtypes of OV, CESC and BLCA, implying an infection risk for SARS-CoV-2 in PAAD and STAD, and for IFN-gamma dominance in OV, CESC and BLCA. To clarify these results derived from database analyses, further verification in a large clinical cohort is essential.

AUTHOR CONTRIBUTIONS
XG and HL and LX designed the study; HL and LX performed the analysis and drafted the manuscript; LC and YH collected the data; LZ and ZY modified the manuscript. All the authors approved this manuscript.

CONFLICTS OF INTEREST
This manuscript has not been published and is not under consideration for publication elsewhere. We have no conflicts of interest to disclose.

Supplementary Tables
Supplementary Table 1