key: cord-0832037-jvnatupd
authors: Deng, Qu; Rasool, Reyaz ur; Russell, Ronnie M.; Natesan, Ramakrishnan; Asangani, Irfan A.
title: Targeting androgen regulation of TMPRSS2 and ACE2 as a therapeutic strategy to combat COVID-19
date: 2021-03-01
journal: iScience
DOI: 10.1016/j.isci.2021.102254
sha: f7d4fe79c19ecf5b5d16937683821c796414f0dc
doc_id: 832037
cord_uid: jvnatupd

Epidemiological data showing increased severity and mortality of COVID-19 in men suggests a potential role for androgen in SARS-CoV-2 infection. Here, we present evidence for the transcriptional regulation of SARS-CoV-2 host cell receptor ACE2 and TMPRSS2 by androgen in mouse and human cells. Additionally, we demonstrate the endogenous interaction between TMPRSS2 and ACE2 in human cells and validate ACE2 as a TMPRSS2 substrate. Further, Camostat – a TMPRSS2 inhibitor, blocked the cleavage of pseudotype SARS-CoV-2 surface Spike without disrupting TMPRSS2-ACE2 interaction. Thus providing evidence for the first time a direct role of TMPRSS2 in priming the SARS-CoV-2 Spike, required for viral fusion to the host cell. Importantly, androgen-deprivation, anti-androgens, or Camostat attenuated the SARS-CoV-2 S-mediated cellular entry. Together, our data provide a strong rationale for clinical evaluations of TMPRSS2 inhibitors, androgen-deprivation therapy/androgen receptor antagonists alone or in combination with antiviral drugs as early as clinically possible to prevent COVID-19 progression.

The ongoing COVID-19 (Coronavirus disease 2019) pandemic caused by Severe Acute

Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is global health crisis (Morens and Fauci, 2020; Wang et al., 2020) . As of January 2021, over 100 million cases of and more than two million deaths have been recorded (https://coronavirus.jhu.edu).

Epidemiological data have shown that males are disproportionally affected, being slightly more likely to be infected than females and accounting for most severely ill cases and higher fatality. This is compounded by older age and comorbidities such as diabetes, cardiovascular diseases, obesity, hypertension and cancer (Williamson et al., 2020) . This sex disparity with respect to increased morbidity and mortality in men suggests a potential role for the male hormone androgen in SARS-CoV-2 infection and host response (Stopsack et al., 2020; Wadman, 2020) .

The host immune response to SARS-CoV-2 potentiates a hyper-inflammatory cytokine storm involving the upregulation of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1 β),

interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein 1-α (MIP1α), which is associated with COVID-19 severity and mortality (Mehta et al., 2020; Moore and June, 2020; Vabret et al., 2020; Zhang et al., 2020) .

Tumor cell-intrinsic and microenvironment associated immune cell response to inflammation promote the development and progression of several types of solid cancer, including prostate cancer (de Bono et al., 2020) . Therefore, it is reasonable to hypothesize that the inflammatory signaling accompanying severe COVID-19 disease could cause cancer progression. Therefore, early intervention of COVID-19 management should be considered in cancer patients who are more susceptible to SARS-CoV-2 infection due to their immunosuppressive state.

SARS-CoV-2 entry into the host cell is mediated by trimers of the transmembrane spike (S) glycoprotein. The S trimer binds to the cellular receptor angiotensin converting enzyme 2 (ACE2) on the surface of target cells and mediates subsequent viral uptake and fusion dependent on processing by host proteases (Hoffmann et al., 2020c) . Several host proteases, including TMPRSS2, cathepsin B and L, and furin have been suggested to promote the entry of SARS-CoV-2; how they process the Spike protein remains to be determined (Coutard et al., 2020;  J o u r n a l P r e -p r o o f Shang et al., 2020; Walls et al., 2020) . In relevant target cells, the cleavage of S by TMPRSS2 activates the protein for membrane fusion via extensive, irreversible conformational changes (Millet and Whittaker, 2015; Walls et al., 2020; Walls et al., 2017) . As a result, SARS-CoV-2 entry into susceptible cells is a complex process that requires the concerted action of ACE2 receptor-binding and TMPRSS2 proteolytic processing of the S protein to promote virus-cell fusion. Despite their well-documented role in SARS-CoV-2 entry, the nature of endogenous ACE2 and TMRPSS2 interactions in host cells is lacking.

TMRPSS2 is a widely studied androgen-regulated gene associated with prostate cancer (Afar et al., 2001) . It contributes to prostate cancer pathogenesis by way of aberrantly driving oncogenic transcription. More than half of all prostate cancers in men of European ancestry harbor a gene fusion that juxtaposes the androgen receptor (AR) regulatory promoter/enhancer elements of Tmprss2 in front of the ETS family transcription factors, most commonly Erg (Tomlins et al., 2005) . Although ERG is not normally regulated by androgen, this somatic gene rearrangement results in oncogenic ERG expression under androgen receptor signaling. Further, TMRPSS2 is known to promote metastatic spread of prostate cancer through HGF activation (Lucas et al., 2014) . ACE2 is a zinc-dependent metalloprotease and is expressed at high levels in the heart, testis, and kidney and at lower levels in various tissue (Gheblawi et al., 2020) . Therefore, identifying molecular mechanisms governing the expression of TMPRSS2 and ACE2 could potentially explain the observed disparity in SARS-CoV-2 infection-associated morbidity and mortality in men.

In this study, we provide evidence for the AR-mediated transcriptional regulation of TMPRSS2 and ACE2 in mice and human prostate and lung cells. Specifically, androgen deprivation in mice by castration, or anti-androgen treatment in vitro led to a reduction in TMPRSS2 and ACE2 transcript and protein levels. Furthermore, TMPRSS2 and ACE2 were found to interact in cis in prostate and lung cells, and the inhibition of TMPRSS2 protease activity by Camostat blocked Spike priming. Finally, androgen-deprivation, anti-androgens, or Camostat treatment attenuated the SARS-CoV-2 pseudotype entry in lung and prostate cells, suggesting that these drug combinations could be effective in attenuating COVID-19 disease progression in men with or without prostate cancer.

J o u r n a l P r e -p r o o f

The poor clinical outcome of COVID-19 in men suggests a potential underlying androgenrelated cause. The role of male sex hormone androgen in regulating the SARS-CoV-2 host cell receptor ACE2 and co-receptor TMPRSS2 has been speculated (La Vignera et al., 2020; Stopsack et al., 2020; Wadman, 2020) . To evaluate the effect of androgen deprivation on TMPRSS2 and ACE2 expression in major tissues that are the primary sites for SARS-CoV-2 infection (Ziegler et al., 2020) , we performed surgical castration in adult male mice (n=3). One

week post-castration, we harvested the lung, small intestine, heart and kidney for qRT-PCR, immunoblotting, and immunohistochemistry analysis. Seminal vesicles and prostate were used as positive controls for systemic androgen deprivation. Tissues from the corresponding mock castrated male (n=3) and female (n=3) served as controls (Fig. 1A) . First, we determined the expression of Tmprss2, Ace2 and Ar in the control tissues. Each was expressed at varying degrees, with Ar and Tmprss2 being the highest in seminal vesicles, and ACE2 in the small intestine ( Supplementary Fig. 1A ). Interestingly, a survey of adult human male tissue mRNA expression via bulk RNA-seq from the GTEx consortium (Consortium et al., 2017 ) demonstrated a similar expression pattern in prostate and small intestine for AR, TMPRSS2, and ACE2

( Supplementary Fig. 1B) , suggesting a potentially identical mechanism of regulation in man and mice. Next, as expected, the Tmprss2 expression was highly androgen-responsive in the seminal vesicles of the castrated males compared to the control males. (Fig. 1B) . Except for the small intestine and the lung which showed a slight downward trend, levels of Tmprss2 in the heart (with very low basal expression), and the kidney did not display any change upon castration.

However, Ace2 expression displayed a significant downregulation in the lung and the small intestine, which was similar to the levels found in female mice, and upregulation in the kidney tissues in the castrated male compared to controls. Remarkably, Ace2 expression was also reduced in the hormone-sensitive seminal vesicles upon castration (Fig. 1B) , suggesting a potential role for androgen in regulating Ace2 expression in these tissues. In concordance with the transcripts, the immunoblot analysis using total protein extracts from tissues demonstrated a reduced TMRPSS2 and ACE2 protein levels in the lung and the small intestine. The reduction in protein levels was much more dramatic than the transcript levels. In contrast, the kidney tissue displayed an increase in the ACE2 protein, but not TMPRSS2, corroborating the transcript data J o u r n a l P r e -p r o o f ( Fig. 1C ). Next, we performed immunohistochemistry to identify the specific cell types in these tissues that display altered expression for TMPRSS2 and ACE2 proteins upon androgen deprivation. In these experiments, DU145 and VCaP prostate cancer xenograft tissues served as a negative control for AR, TMPRSS2, and ACE2 respectively, which showed a clear lack of staining (Supplementary Fig. 2A ). As expected, based on the transcript data, reduced staining for TMPRSS2, ACE2, and AR protein in hormone-sensitive seminal vesicles was observed in the castrated group ( Supplementary Fig. 2B ). Further, the hormone-responsive prostate luminal epithelial cells showed reduced staining for AR and TMPRSS2 in response to castration (Fig.   1D ). We also observed a small minority population of epithelial cells <5% staining for ACE2 on the apical surface of the prostate epithelium, which was absent in the castrated group. The identical minimal co-expression pattern of ACE2 and TMPRSS2 was also identified in human prostate tissues, where club cells that constitute a mere 0.07% of total prostate epithelial cells and resemble club cells in the lung were double-positive (Henry et al., 2018; Montoro et al., 2018; Song et al., 2020) . We observed no morphological changes in response to castration in lung, small intestine and kidney tissue through H&E staining. Similar to transcripts, the AR, ACE2, and TMPRSS2 proteins were detected in all of the tested tissue types at varying degrees ( Fig. 1E) . A strong staining for TMPRSS2 was detected exclusively in the lung respiratory bronchiole epithelium, with minimal staining of type II alveolar cells, which is consistent with the expression pattern observed in human tissue (Stopsack et al., 2020) . The staining intensity, especially in type II alveolar cells, was reduced in the castrated group. The ACE2 staining could be observed in the lung parenchyma, both type I and type II alveolar cells and the bronchiole epithelium apical surface of the castrated males showed relatively lower expression ( Fig. 1E and Supplementary Fig. 2C ). Next, the small intestine mucosa from the control group showed positive cytoplasmic and nuclear AR staining, which were reduced in the castrated group.

TMPRSS2 displayed high staining in the crypt and lower portion of the villi with the exception in the goblet cells, and the staining gradually diminished on the top of the villi. ACE2 was mainly present on the apical surface of the enterocytes on the top part of the villi. These expression patterns mirror data from human small intestinal tissue, especially ileum and jejunum (Hamming et al., 2004; Ziegler et al., 2020) . The abundance of double-positive cells was largely reduced in the castrated male tissue due to the reduction in both ACE2 and TMPRSS2 levels.

Concerning the kidney, the AR staining was detected in the tubular cells, which was reduced J o u r n a l P r e -p r o o f upon castration. TMPRSS2 showed positivity mainly in the proximal tubules compared to other cells, which was reduced in the castrated group (Muus et al., 2020) . Finally, a homogenous ACE2 expression was detected across kidney tissue on the apical surface of the tubular cells. In line with the kidney's role in the renin-angiotensin-aldosterone system, the ACE2 intensity was strongly elevated in the castrated males responding to reduced blood pressure brought out by androgen deprivation (Dubey et al., 2002) . Together, these analyses clearly demonstrate that the androgen deprivation has an effect on the expression of TMPRSS2 and ACE2, particularly in the lung, small intestine, and kidney. Though there was a discrepancy concerning transcript and protein levels of TMPRSS2 in the tested tissues -particularly in the kidney, post-transcriptional regulation of TMPRSS2 by androgen regulated microRNA could not be ruled out (Nersisyan et al., 2020) .

The observation that systemic androgen deprivation affects the expression of TMPRSS2 and ACE2 in hormone-sensitive tissues such as prostate and seminal vesicles, but also in major organs such as the lungs, small intestine, and kidney led us to investigate the direct role of AR in the transcriptional regulation of these two critical genes beyond the prostate. Although the upstream enhancer of Tmprss2 is a well-known target for AR (Asangani et al., 2014) , we sought to identify whether regulatory elements of Ace2 are also occupied by AR. We analyzed publicly available AR ChIP-seq data for AR-binding sites within 50 kb of the transcription start sites (TSSs) of the Tmprss2 and Ace2 genes in mouse prostate, comparing castration and castration followed by testosterone injection for 3 days (Pihlajamaa et al., 2014) . As expected, multiple AR peaks were observed in the upstream of the Tmprss22 TSS in the testosterone-treated prostate, which was also present in DHT (dihydrotestosterone) treated VCaP and LNCaP human prostate cell lines (Fig. 2a) . Interestingly, the upstream region of ACE2 also showed AR binding in mouse as well as in human prostate cells ( Fig Interestingly, androgen induced AR dependent upregulation of TMPRSS2 has been demonstrated in other lung cancer cell lines (Mikkonen et al., 2010) . These data clearly demonstrate a potential role of AR in regulating SARS-CoV-2 receptor and co-receptor in prostate and lung cells.

Having identified the androgen dependency of TMPRSS2 and ACE2 expressions in vivo in mice and in vitro in human cell lines, we turned our attention to their function concerning SARS-CoV-2 Spike (SARS-2-S) priming. Though TMPRSS2 is implicated in priming of SARS-2-S protein (Hoffmann et al., 2020c) , it is unclear whether this occurs in association with ACE2. Further, TMPRSS2-mediated enhancement of SARS virus entry has been shown to accompany ACE2

cleavage (Shulla et al., 2011) . Therefore, we studied whether TMPRSS2 and ACE2 physically associate in an endogenous setting and examined the effect of TMPRSS2 protease inhibition on J o u r n a l P r e -p r o o f their interaction and cleavage of ACE2. TMPRSS2 is composed of a cytoplasmic region (aa 1-84), a transmembrane region (aa 85-105), and an extracellular region (aa 106-492). The latter is composed of three domains: the LDLRA (LDL receptor class A) domain (residues 112-149)that forms a binding site for calcium, the SRCR (scavenger receptor cysteine-rich) domain (aa 149-242) -involved in the binding to other cell surface or extracellular proteins, and the Peptidase S1 domain (aa 256-489), which contains the protease active site: residues H296, D345, and S441 (Fig. 4A ). An auto-cleavage site at aa 255-256 has been shown to allow shedding of the extracellular region of TMPRSS2 (Afar et al., 2001) . The ACE2 protein is 805 aa long and is composed of a short cytoplasmic region, a transmembrane region, and an extracellular Nterminal region that contain zinc metallopeptidase domain (Fig. 3A ). TMPRSS2 mediated cleavage of ACE2 at its N-terminus (residues 697 to 716) was shown to be required for ACE2 to interact with SARS-S protein in 293T overexpression system (Heurich et al., 2014; Shulla et al., 2011) . Our experiments show that overexpression of TMPRSS2 in HEK293-T cells that stably express ACE2 led to ACE2 cleavage, resulting in reduced levels of the ∼120 kDa full-length form and increased levels of a ∼20 kDa C-terminal fragment ( Fig. 4B ). These data demonstrate for the first time an endogenous association between TMPRSS2 and ACE2 in prostate and lung cells.

Next, we addressed the role of TMPRSS2 on priming of SARS-CoV-2 Spike (SARS-2-S) -a heavily glycosylated transmembrane protein (Cai et al., 2020; Ke et al., 2020) (Fig. 4A) . The unprocessed Spike S0 protein encoded by the SARS-CoV-2 genome is 1,273 amino acid long. It is incorporated into the viral membrane and facilitates viral entry into target cells. Similar to SARS CoV and MERS CoV Spike proteins, SARS-CoV-2 Spike consists of a proprotein convertase (PPC) motif at the S1/S2 boundary cleaved by furin protease and TMPRSS2 into two fragments -the receptor-binding fragment S1 and the membrane fusion fragment S2 (Hoffmann et al., 2020b; Hoffmann et al., 2020c; Iwata-Yoshikawa et al., 2019; Shang et al., 2020; Shulla et al., 2011; Zhou et al., 2015) . Though the role of TMPRSS2 in SARS-2-S priming has been suggested (Hoffmann et al., 2020c) , direct evidence demonstrating the cleaving of SARS-2-S by endogenous TMPRSS2 is lacking. Therefore, we performed experiments to specifically address the role of ectopically expressed or endogenous TMPRSS2 in SARS-2-S activation. Transfection of codon optimized plasmid encoding SARS-2-S protein and 2xStrep.tag (Gordon et al., 2020) in HEK293-T showed a distinct ∼180 kDa, and a ∼280 kDa band which is likely the differentially glycosylated S0 protein. The two other bands at ∼120 kDa and a ∼60 kDa could be the nonglycosylated fragments (Fig. 4B ). The S1 fragment could not be detected in the immunoblots as the streptavidin-tag is located at the C-terminal end of the constructs. The co-transfection with TMPRSS2 plasmid led to cleavage of S0 into distinct bands corresponding to S2, and S2' (Fig.   4B ). Interestingly, the ∼280 kDa band and ∼120 kDa was fully processed by TMPRSS2, suggesting the S1/S2 site on glycosylated and non-glycosylated S0 protein could be equally J o u r n a l P r e -p r o o f 4C and Supplementary Fig. 5A-B ). Additionally, we tested and found that HEK293-T ACE2 cells have superior uptake of HIV pseudotype SARS-2-S compared to HEK293-T cells, confirming ACE2 as the major receptor SARS-CoV-2 ( Supplementary Fig. 5C ). In agreement with a recent study that demonstrated the existence of S mostly in the closed uncleaved prefusion confirmation on the authentic SARS-CoV-2 (Turonova et al., 2020), the purified pseudotype SARS-2-S virus spinoculation followed by immunoblotting displayed both glycosylated ∼180 kDa and non-glycosylated ∼120 kDa full-length S0 protein (Fig. 4D) with LNCaP and Calu-3 cells ( Fig. 4F and 4G ). These data clearly demonstrate the crucial role played by TMPRSS2 in SARS-CoV-2 viral fusion to the host cell by Spike protein priming (Fig.   4H ).

After identifying the role of androgen in regulating TMRPSS2 and ACE2 expression, and TMPRSS2 mediated SARS-2-S priming, we asked whether therapeutic intervention targeting AR and TMPRSS2 protease function could affect SARS-CoV-2 infection in prostate and lung cells. Replication-defective virus particles bearing SARS-2-S proteins faithfully reflect critical J o u r n a l P r e -p r o o f aspects of SARS-CoV-2 host cell entry (Hoffmann et al., 2020c) . Therefore, we employed HIV pseudotypes bearing SARS-2-S and nano-luciferase reporter to test the efficacy of Camostat and AR directed therapies in blocking cell entry (Fig. 5A) . We first asked whether androgen deprivation could affect pseudotype virus entry by reducing TMRPSS2 and ACE2 (Fig.2) .

LNCaP cells grown in the androgen proficient condition were readily susceptible to the entry driven by SARS-2-S (Fig. 5B) . However, cells grown under steroid-deprived condition (CSScharcoal stripped serum) demonstrated a significant reduction in the entry of pseudotype virus.

Interestingly, Camostat treatment efficiently blocked the SARS-2-S mediated entry only in the androgen proficient condition, suggesting the presence of TMPRSS2 as a requirement for its potency. Next, we treated LNCaP cells grown in normal growth condition with anti-androgen enzalutamide, AR degrader ARD-69, or Camostat and observed a significant reduction in SARS-2-S driven entry compared to the DMSO control. Notably, the combination of Camostat with enzalutamide or ARD-69 was more efficacious in blocking the entry than single drug (Fig. 5C ).

Since lungs are the primary target of SARS-CoV-2, we tested whether androgen deprivation, enzalutamide, ARD-69 and Camostat could block SARS-2-S mediated entry in AR positive H460 cells. Similar to LNCaP cells, pseudovirus entry was significantly reduced in the H460 cells grown in the steroid-deprived condition, enzalutamide, and ARD-69 alone or in combination with Camostat compared to the control ( Fig. 5D and 5E ). As expected, compared to Camostat, the enzalutamide or AR degrader treatment in AR-negative Calu-3 cells did not block the SARS-2-S mediated entry (Fig. 5F ). Importantly, unlike SARS-2-S, HIV pseudotypes bearing VSV-G did not display any significant difference in cell entry under these condition in all three tested cell lines ( Supplementary Fig. 6 ). These results indicate that the treatment of ARpositive prostate and lung cells with AR directed therapies in combination with TMPRSS2

inhibitor efficiently block SARS-2-S mediated viral entry.

Early intervention of SARS-CoV-2 infection could prevent cytokine storm mediated progression to severe pneumonia and multi-organ failure in COVID-19 patients (Mehta et al., 2020) .

Although the source of the cytokine storm that causes multi-organ dysfunction is not yet clear, Growing evidence suggests cancer patients are susceptible to severe form of COVID-19 mainly due to their immunosuppressive state and co-existing medical conditions (Dai et al., 2020; Lee et al., 2020; Tang and Hu, 2020) . In general, patients with metastatic cancers infected with SARS-CoV-2 have had poorer outcomes including death, admission to the intensive care unit, requiring mechanical ventilation, and severe symptoms (Dai et al., 2020) . As the development of vaccines and anti-viral drugs against the pandemic causing SARS-CoV-2 are proceeding rapidly,

Camostat mesilate along with androgen regulation of TMPRSS2 and ACE2, as a means to inhibit J o u r n a l P r e -p r o o f SARS-CoV-2 cell entry (Fig. 5G) , and thus, the infection represents a potential strategy in treating COVID-19 in these high-risk population.

Androgen plays a vital role in the human immune response, which we did not address in this paper. Research aimed at characterizing the androgen axis in context with SARS-CoV-2 infection will continue in the future.

Lead Contact: Further information and requests for resources should be directed to and will be fulfilled by the Lead Contact, Irfan A. Asangani (asangani@upenn.edu).

Materials Availability: This study did not generate new unique reagents.

Data and Code Availability: No large datasets were generated within this study. Original data can be made available from the lead contact on request.

All methods can be found in the accompanying Transparent Methods supplemental file.

We protein that has S1 and S2 segments involved in virus attachment and fusion, respectively.

Segments of S1 include RBD, receptor-binding domain; S1/S2 cleavage site; S2 has an S2' cleavage site. Tree-like symbol denotes glycans, and the C terminus contains 2x Strep-tag. The Spike S, ACE2 and TMPRSS2 leading to cleavage of Spike S into S1, S2 and S2' segments.

S0/S1 continues to bind tightly through its RBD to ACE2 that is in complex with TMPRSS2, J o u r n a l P r e -p r o o f whereas upon cleavage the S2 and S2' gets dissociated from the TMPRSS2-ACE2 complex.

Also, see Supplementary Figure 5 . • TMPRSS2 interacts with ACE2 in prostate and lung cells • Camostat blocks TMPRSS2 mediated cleavage of SARS-Cov-2 Spike • Androgen-deprivation or AR-antagonists attenuates SARS-CoV-2 Spike mediated cell entry

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