Impacts of transmembrane serine protease 4 expression on susceptibility to severe acute respiratory syndrome coronavirus 2

To the Editor: Transmembrane serine protease 4 (TMPRSS4; OMIM: 606565), which encodes a member of the serine protease family, is involved in various biological processes related to diseases such as cancer and pediatric neurodegenerative disorders.[1,2] Overexpression of this gene (NM_019894.4) was first identified in pancreatic carcinoma with metastasis and invasion,[3] suggesting its potential as a therapeutic target and prognostic marker.[4] Besides, TMPRSS4 has been found to promote severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry into host cells by cleaving SARS-CoV-2 spike glycoproteins (S protein) into an active glycoprotein that facilitates coronavirus infection.[5]

Coronavirus disease 2019 (COVID-19) is caused by infection with SARS-CoV-2. Cancer patients with COVID-19 have worse outcomes. Research found that abnormally high expression of proteins, which facilitate SARS-CoV-2 infection, such as TMPRSS4, caused a worse prognosis of the COVID-19.[6] However, TMPRSS4 expression in normal and cancer tissues has not been fully investigated, and it is also unknown whether small molecules derived from Chinese medicine can suppress TMPRSS4 expression. In this study, we aim to analyze TMPRSS4 expression, isoform information, and the impact of TMPRSS4 on COVID-19 susceptibility in healthy and cancer individuals, as well as to monitor TMPRSS4 expression after the treatment of cordycepin (CD), thymoquinone (TQ) and N6, N6-dimethyladenosine (m62A) in cancer cells.

Bioinformatics analysis [Supplementary File, https://links.lww.com/CM9/B287] showed that TMPRSS4 is highly conserved across different species, such as human, chimpanzee, Rhesus monkey, dog, cow, mouse, rat, chicken, frog, zebrafish, and fruit fly [Supplementary Figure 1A, https://links.lww.com/CM9/B287]. High conservation implies that TMPRSS4, which is similar to TMPRSS2 and other receptors in different species, has the potential to cleave the receptor-binding domain of the S-protein of SARS-CoV-2. TMPRSS4 expression at both the transcriptional and translational level in human organs and tissues was presented in Supplementary Figure 1B, https://links.lww.com/CM9/B287, which shows that TMPRSS4 messenger ribonucleic acid (mRNA) was primarily expressed in the proximal digestive tract, kidney, urinary bladder, and gastrointestinal tract, then in the bone marrow, lymphoid tissues, reproductive organs, liver, gallbladder, and skin, whereas the distribution of TMPRSS4 protein was significantly distinct from the mRNA expression patterns. With the exception of the eyes, TMPRSS4 protein expression was generally observed in all organs and tissues, including the gastrointestinal tract and lungs, and the top four tissues for TMPRSS4 mRNA expression were the esophagus (proximal digestive tract), urinary bladder (kidney and urinary bladder), colon, and rectum (gastrointestinal tract) [Supplementary Figure 1C, https://links.lww.com/CM9/B287]. Regarding the protein expression according to the immunohistochemistry scores, we found that 36 organs and tissues had moderate expression of TMPRSS4, while low expression was noted in the cerebellum, salivary gland, liver, kidney, ovary, smooth muscle, and lymph nodes [Supplementary Figure 1D, https://links.lww.com/CM9/B287]. Furthermore, mRNA levels of single-cell type specificity indicated that TMPRSS4 was dominantly expressed in urothelial cells, distal enterocytes, airway basal cells, club cells, and squamous epithelial cells [Supplementary Figure 1E, https://links.lww.com/CM9/B287]. TMPRSS4 mRNA expression in the brain and immune cells was very low but remained at detectable levels in the cerebellum (1.8 normalized protein-coding transcripts per million [nTPM]) [Supplementary Figure 1F, https://links.lww.com/CM9/B287] and in the basophils (2.1 nPTM) [Supplementary Figure 1G, https://links.lww.com/CM9/B287].

Furthermore, according to the Human Protein Atlas (HPA) datasets analysis, in cancer tissues, TMPRSS4 protein showed weak to moderate expression; the TMPRSS4 protein showed strong expression in some cases of seminomas according to the immunohistochemical results in the HPA dataset HPA0006238. Above analysis also showed that TMPRSS4 mRNA exhibited the highest expression in pancreatic adenocarcinoma (PAAD), which is consistent with a previous report.[3] Moreover, the difference of TMPRSS4 mRNA expression in different cancer tissues and corresponding normal tissues was compared by Gene Expression Profiling Interactive Analysis 2 (GEPIA2). We found that cervical squamous cell carcinoma, endocervical adenocarcinoma, colon adenocarcinoma, lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), ovarian serous cystadenocarcinoma, PAAD, rectum adenocarcinoma, stomach adenocarcinoma, thyroid carcinoma, uterine corpus endometrial carcinoma, and uterine carcinosarcoma had significantly higher TMPRSS4 expression compared with corresponding normal tissues [Supplementary Figure 2A, https://links.lww.com/CM9/B287]; only kidney chromophobe, kidney renal papillary cell carcinoma (KIRP), kidney renal clear-cell carcinoma, acute myeloid leukemia, skin cutaneous melanoma, and testicular germ cell tumors showed significantly lower TMPRSS4 expression when compared with the corresponding normal tissues [Supplementary Figure 2A, https://links.lww.com/CM9/B287]. Herein, TMPRSS4 expression was significantly higher in the respiratory tract, particularly in LUAD and LUSC, than in the corresponding normal lung tissues. Further exploration of the prognostic value of TMPRSS4 revealed that higher expression of TMPRSS4 was associated with shorter overall survival (OS) in KIRP and PAAD [Supplementary Figure 2B, 2C, https://links.lww.com/CM9/B287], but with longer OS in bladder urothelial carcinoma [Supplementary Figure 2D, https://links.lww.com/CM9/B287].

Additionally, two critical organs that are subject to SARS-CoV-2 attack are the lungs and pancreas, where tissue expression of TMPRSS4/TMPRSS2 was high. TMPRSS2 is the second identified entry protein/receptor for SARS-CoV-2 attack; both TMPRSS2 and TMPRSS4 are members of type II transmembrane serine proteases. Thus, we compared TMPRSS4 and TMPRSS2 expression in lung cancer and PAAD tissues with corresponding normal tissues using GEPIA 2 in The Cancer Genome Atlas (TCGA) database. We noticed that TMPRSS4 exhibited high expression in some cancer tissues including LUSC and PAAD compared with the corresponding normal tissues. Conversely, TMPRSS2 had comparable levels in both cancer and normal tissues. TMPRSS4 expression in normal lung and pancreatic tissues is much lower than that of TMPRSS2 [Supplementary Figures 3A and 3B, https://links.lww.com/CM9/B287]. According to these expression patterns, we concluded that TMPRSS4 may play more important roles than TMPRSS2 in SARS-CoV-2 entry into lung cancer and PAAD patients, while TMPRSS2 may be primarily responsible for SARS-CoV-2 attack in healthy people.

Angiotensin-converting enzyme 2 (ACE2) is the first identified entry protein/receptor for SARS-CoV-2 attack. Different isoforms of ACE2 might have differential contributions to host susceptibility to SARS-CoV-2 entry. We thus also analyzed TMPRSS4 isoform prevalence and its structures across pan-cancers by GEPIA2. More than 21 isoforms with differential expression were identified in pan-cancers [Supplementary Figure 4A, https://links.lww.com/CM9/B287]. Except for very low or no expression of isoforms ENST00000610964.4 (TMPRSS4–201), ENST00000522151.5 (TMPRSS4–011), and ENST00000518413.2 (TMPRSS4–022), the other 18 TMPRSS4 isoforms were detected in 33 cancer types. Fourteen cancers expressed more than one isoform [Supplementary Figure 4A, https://links.lww.com/CM9/B287]. Among them, the isoform expression of ENST00000437212.7 (TMPRSS4–004) was the highest among 14 cancers, followed by ENST00000524218.1 (TMPRSS4–017), with very few or none of the other 19 isoforms expressed [Supplementary Figure 4B, https://links.lww.com/CM9/B287]. The structures of TMPRSS4 isoforms ENST00000437212.7 (TMPRSS4–004) exhibited the same structure as reported previously, with 437 amino acids, and have both SRCR_2 and trypsin domains [Supplementary Figure 4C, https://links.lww.com/CM9/B287], which are not in other isoforms, demonstrating the functional roles of this protein in tumorigenesis and SARS-CoV-2 entry into cancer patients. Considering its highly elevated expression and the poor survival of PAAD patients, isoform ENST00000437212.7 (TMPRSS4–004) may have roles in both tumorigenesis and SARS-CoV-2 entry in PAAD patients.

Notably, CD, a natural product derived from the traditional Chinese medicine fungus Cordyceps militaris showing anti-cancer roles in vitro and in vivo, was tested for its inhibitory effects on TMPRSS4 expression in cancer cell lines [Supplementary File, https://links.lww.com/CM9/B287]. Western blotting and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) [Supplementary File, https://links.lww.com/CM9/B287] results showed that CD suppressed the expression of both TMPRSS4 protein and mRNA in a dose-dependent manner in H1975 lung cancer cells [Figures 1A, 1B], MCF7 breast cancer cells [Figures 1C, 1D], and 22RV1 prostate cancer cells [Figures 1E, 1F].

F1Figure 1:

CD, TQ, and N6,N6-dimethyladenosine (m62A) suppress TMPRSS4 expression in various cancer cell lines. (A) Western blotting results showed that CD suppressed TMPRSS4 expression in H1975 cells. (B) RT-PCR results showed that CD suppressed TMPRSS4 expression in H1975 cells. (C) Western blotting results showed that CD suppressed TMPRSS4 expression in MCF7 cells. (D) RT-PCR results showed that CD suppressed TMPRSS4 expression in MCF7 cells. (E) Western blotting results showed that CD suppressed TMPRSS4 expression in 22RV1 cells. (F) RT-PCR results showed that CD suppressed TMPRSS4 expression in 22RV1 cells. (G) Western blotting results showed that TQ suppressed TMPRSS4 expression in 22RV1 cells. (H) RT-PCR results showed that TQ suppressed TMPRSS4 expression in 22RV1 cells. (I) Western blotting results showed that m62A suppressed TMPRSS4 expression in H460 cells. (J) RT-PCR results showed that m62A suppressed TMPRSS4 expression in H460 cells. ACTB: Actin beta; CD: Cordycepin; m62A: N6, N6-dimethyladenosine; HSP70: Heat shock protein 70; RT-PCR: Reverse transcription-polymerase chain reaction; TQ: Thymoquinone; TMPRSS: Transmembrane serine protease.

Moreover, the effects of TQ, a component of Nigella sativa seeds showing anti-cancer roles in vitro and in vivo, on TMPRSS4 expression was examined by Western blotting and RT-PCR [Supplementary File, https://links.lww.com/CM9/B287], and the results showed that it suppressed the expression of both TMPRSS4 protein and mRNA in a dose-dependent manner in 22RV1 prostate cancer cells [Figures 1G, 1H].

Another nucleoside derivative of CD, m62A, was examined for its inhibitory effect on TMPRSS4 expression by Western blotting and RT-PCR [Supplementary File, https://links.lww.com/CM9/B287]. The results showed that m62A suppressed the expression of both TMPRSS4 protein and mRNA in a dose-dependent manner in H460 lung cancer cells [Figures 1I, 1J]. Thus, both CD and m62A, and the small molecule TQ may have therapeutic potential as anti-SARS-CoV-2 agents by suppressing TMPRSS4 expression.

In conclusion, our work revealed the expression and distribution of TMPRSS4 in various tissues, and implied that the expression of TMPRSS4 has specific significance for prognosis across different cancer types. TMPRSS4 may play an important role in SARS-CoV-2 attack in lung cancer and PAAD patients. Small molecules derived from traditional Chinese medicine, could be applied in the development of anti-SARS-CoV-2 drugs as well as anti-cancer agents by suppressing TMPRSS4 expression. Our study thus highlights the value of targeting TMPRSS4 as an alternative therapeutic strategy to combat COVID-19.

Acknowledgements

The authors thank all the people from the Research Center for Preclinical Medicine, Southwest Medical University.

Funding

This work was supported by the Foundation of Southwest Medical University (Nos. 2021ZKMS004 and 2021ZKQN109), the Research Foundation of the Science and Technology Department of Sichuan Province (No. 2022NSFSC0737), in part by the Research Foundation of Luzhou City (No. 2021-SYF-37), and the National Natural Science Foundation of China (Nos. 81672887 and 82073263).

Conflicts of interest

None.

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