Prostate adenocarcinoma and COVID‐19: The possible impacts of TMPRSS2 expressions in susceptibility to SARS‐CoV‐2

Abstract TMPRSS2 (OMIM: 602060) is a cellular protease involved in many physiological and pathological processes, and it facilitates entry of viruses such as SARS‐CoV‐2 into host cells. It is important to predict the prostate's susceptibility to SARS‐CoV‐2 infection in cancer patients and the disease outcome by assessing TMPRSS2 expression in cancer tissues. In this study, we conducted the expression profiles of the TMPRSS2 gene for COVID‐19 in different normal tissues and PRAD (prostate adenocarcinoma) tumour tissues. TMPRSS2 is highly expressed in normal tissues including the small intestine, prostate, pancreas, salivary gland, colon, stomach, seminal vesicle and lung, and is increased in PRAD tissues, indicating that SARS‐CoV‐2 might attack not only the lungs and other normal organs, but also in PRAD cancer tissues. Hypomethylation of TMPRSS2 promoter may not be the mechanism for TMPRSS2 overexpression in PRAD tissues and PRAD pathogenesis. TMPRSS2 expresses eleven isoforms in PRAD tissues, with the TMPRSS2‐001 isoform expressed highest and followed by TMPRSS2‐201. Further isoform structures prediction showed that these two highly expressed isoforms have both SRCR_2 and Trypsin (Tryp_SPc) domains, which may be essential for TMPRSS2 functional roles for tumorigenesis and entry for SARS‐CoV‐2 in PRAD patients. Analyses of functional annotation and enrichment in TMPRSS2 showed that TMPRSS2 is mostly enriched in regulation of viral entry into host cells, protein processing and serine‐type peptidase activity. TMPRSS2 is also associated with prostate gland cancer cell expression, different complex(es) formation, human influenza and carcinoma, pathways in prostate cancer, influenza A, and transcriptional misregulation in cancer. Altogether, even though high expression of TMPRSS2 may not be favourable for PRAD patient's survival, increased expression in these patients should play roles in susceptibility of the SARS‐CoV‐2 infection and clinical severity for COVID‐19, highlighting the value of protective actions of PRAD cases by targeting or androgen‐mediated therapeutic strategies in the COVID‐19 pandemic.

the lungs and other normal organs, but also in PRAD cancer tissues. Hypomethylation of TMPRSS2 promoter may not be the mechanism for TMPRSS2 overexpression in PRAD tissues and PRAD pathogenesis. TMPRSS2 expresses eleven isoforms in PRAD tissues, with the TMPRSS2-001 isoform expressed highest and followed by TMPRSS2-201. Further isoform structures prediction showed that these two highly expressed isoforms have both SRCR_2 and Trypsin (Tryp_SPc) domains, which may be essential for TMPRSS2 functional roles for tumorigenesis and entry for SARS-CoV-2 in PRAD patients. Analyses of functional annotation and enrichment in TMPRSS2 showed that TMPRSS2 is mostly enriched in regulation of viral entry into host cells, protein processing and serine-type peptidase activity. TMPRSS2 is also associated with prostate gland cancer cell expression, different complex(es) formation, human influenza and carcinoma, pathways in prostate cancer, influenza A, and transcriptional misregulation in cancer. Altogether, even though high expression of TMPRSS2 may not be favourable for PRAD patient's survival, increased expression in these patients should play roles in susceptibility of the SARS-CoV-2 infection and clinical severity for COVID-19, highlighting the value of protective actions of PRAD cases by targeting or androgen-mediated therapeutic strategies in the COVID-19 pandemic.

| INTRODUC TI ON
Transmembrane serine protease 2 (TMPRSS2, OMIM: 602060), cytogenetic located at 21q22.3, was first identified by exon trapping in 1997, which encodes a 492 amino acids multimeric protein with a molecular mass 53 859 Da containing a serine protease domain. 1 In prostate cancer tissues, Tomlins et al in 2005 identified recurrent gene fusions at the TMPRSS2 5′ UTR (untranslated region) to ETV1 or ERG with an outlier expression that drives cancer progression, suggesting oncogenic roles in prostate cancer. 2 The cellular protease TMPRSS2 protein is highly expressed in secretory epithelial cells of the prostate, and its expression is androgen-induced. As a member of serine protease family, TMPRSS2 is involved in many pathological and physiological processes, [3][4][5] and also facilitates entry of viruses, including the human coronaviruses SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), SARS-CoV (severe acute respiratory syndrome coronavirus), HCoV-229E (human coronavirus-229E), and MERS-CoV (Middle East respiratory syndrome coronavirus), into host cells by cleaving and activating viral envelope glycoproteins, or proteolytical cleaving ACE2 (angiotensin-converting enzyme 2) receptor (OMIM: 300332) for viral uptake. [6][7][8][9] Since December 2019, the coronavirus disease 2019 (COVID- 19) has rapidly spread worldwide, caused a global threat and the number of cases is rising worldwide. 10

| Promoter methylation analysis for TMPRSS2
The protein expression and promoter methylation status of

| Survival analysis for PRAD in TMPRSS2 expressions
Two expression groups based on the value of fragments per kilobase of exon model per million reads mapped (FPKM) in each gene

| Analysis for functional enrichment
The data of GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway of the co-expressed genes were analysed via the Enrichr database (https://maayanlab.cloud/Enrichr/enrich?datas et=5df6eaa47475293efe5b1514669a05bc#). 29 The P-value < .05 was set as a cut-off criterion. The GEPIA 2 database was used to provide a group of genes with a similar expression pattern between TMPRSS2 and PRAD based on the TCGA-PRAD cohort data.

| Determination of TMPRSS2 conservation and expression in normal tissues
Homologs of the TMPRSS2 protein showed that it is highly conserved in different species, including chimpanzee, Rhesus monkey, dog, cow, F I G U R E 1 Homologs of the TMPRSS2 proteins and its expression in normal tissues and cells. A, Conservation for TMPRSS2 in eleven of different species. B, TMPRSS2 mRNA expression in normal tissues. RNA expression overview shows RNA of consensus NX (Normalized eXpression) levels from 55 types of tissues and 6 types of blood cells, created by combining three different transcriptomics sources: RNAseq data from HPA, RNA-seq data from GTEx and CAGE data from FANTOM5. Colour-coding is based on tissue groups with common functional features. HPA, Human Protein Atlas. GTEx, Genotype-Tissue Expression mouse, rat, chicken, zebra fish, C. elegans, and frog, with a trypsin-like serine protease domain (Tryp_SPc, cd00190) ( Figure 1A). Trypsin-like serine protease is synthesized from inactive precursor zymogens by cleavage to generate their active forms. These suggest that TMPRSS2 from these different animals would potentially have enzymatic activity, making these species SARS-CoV-2's probable natural hosts.
The expression profiles for TMPRSS2 mRNA in humans were conducted from the data of RNA-sequencing in the indicated fiftyfive types of tissues and six types of blood cells that are the consensus dataset from HPA, GTEx and FANTOM5. The RPKM values for TMPRSS2 expression in the small intestine were found to be highest at 75.6, followed by the prostate (68.2), pancreas (64.5), salivary gland (52.3), colon (38.7), stomach (36.7), and lungs is ninth highest expression (20.7). The hypothalamus was found to be lowest with approximately 0.1 ( Figure 1B). No expression was found in 6 blood cell types.
Thus, these results demonstrated the biased expression profiles for TMPRSS2 mRNA in the small intestine, prostate, lung and other tissues.

| Expression analysis results of the TMPRSS2 gene in prostate adenocarcinoma (PRAD)
Gene expression profile for TMPRSS2 in 32 different tumour tissues and their corresponding normal tissues (TCGA normal and GTEx data) F I G U R E 2 TMPRSS2 expression and its promoter methylation status in tumour tissues of prostate adenocarcinoma (PRAD) and corresponding normal tissues. A, Expression profile for TMPRSS2 in 32 different tumour tissues and their corresponding normal tissues (TCGA normal and GTEx data). Tissue-wise expression using profiles. B, Expression profile for TMPRSS2 in PRAD tumour tissues and the corresponding normal tissues (TCGA normal and GTEx data) (*: P <.01). Tissue-wise expression using box plots. C, The promoter methylation status for the regulating TMPRSS2 expression from PRAD. D, Pearson analysis for correlation between the mRNA expression and the methylation status for TMPRSS2 from PRAD. E, Spearman analysis for correlation between the mRNA expression and the methylation status for the TMPRSS2 gene from PRAD revealed six significantly up-regulated (Figure 2A, in red colours) and six down-regulated (Figure 2A, in green colours) in different types of adenocarcinomas. Importantly, both prostate adenocarcinoma and corresponding normal prostate tissues were highly expressed ( Figure 2A, arrow). Further analysis in PRAD found that TMPRSS2 expression is significantly up-regulated ( Figure 2B,P < .01).
To further know whether methylation modification affects TMPRSS2 expression, the DNMIVD database was used to determine the promoter methylation status for TMPRSS2 in PRAD. However, the promoter methylation statuses for the TMPRSS2 in PRAD tissues were slightly increased in comparison with those of normal tissues ( Figure 2C). Furthermore, the analysis of Spearman and Pearson correlations revealed a negative correlation between the TMPRSS2 mRNA expression and its promoter methylation status for PRAD tissues ( Figure 2D and F). Thus, promoter methylation of TMPRSS2 may not be the molecular mechanism for TMPRSS2 overexpression in PRAD tumours and PRAD's pathogenesis.

| Analysis of isoform usage and isoform structures for TMPRSS2
Different isoforms of SARS-CoV-2 receptors or entry proteins, for example isoforms of ACE2 expressed in the airway epithelium, may differentially contribute to host susceptibility to SARS-CoV-2 infection. 30

| Survival analysis for PRAD patients based on TMPRSS2 expression
Given that the study focused on the expression of TMPRSS2 and showed that the expression of TMPRSS2 is higher in both normal and cancerous tissues from PRAD, clinical relationship between TMPRSS2 expression and survival outcomes was also examined. The GENT2 databases were used to assess the TCGA-COAD cohort data and plotted Kaplan-Meier curves. The results are shown in Figure 4.
From Figure 4, we found that high expression of TMPRSS2 is not correlated with long survival in either overall survival ( Figure 4A,P = . 38) or disease-free survival states ( Figure 4B,P = .65). Thus, high expression of TMPRSS2 may not be favourable for PRAD patient's survival.

| Function analysis of co-expressed genes for TMPRSS2 in prostate cancer
Analysis for co-expression in GEPIA 2 database gave a total of 100 associated genes for TMPRSS2 in PRAD (Supplementary Table S1).
The GO analysis results from the Enrichr database are shown in

| D ISCUSS I ON
Highly expressed entry proteins for SARS-CoV-2 may play critical roles for viral infection. 22,31-33 TMPRSS2-expressing cell line has been reported to be highly susceptible to SARS-CoV-2 infection. 34

E TH I C S A PPROVA L
The study has the Ethical Committee approval granted by the Southwest Medical University. This article does not contain any studies with human participants performed by any of the authors.

CO N FLI C T S O F I NTE R E S T
None.