Enzalutamide, a prostate cancer therapeutic, downregulates TMPRSS2 in lung and reduces cellular entry of SARS-CoV-2


 The COVID-19 pandemic, caused by the novel human coronavirus SARS-CoV-2 coronavirus, attacks various organs but most destructively the lung. It has been shown that SARS-CoV-2 entry into lung cells requires two host cell surface proteins: ACE2 and TMPRSS2. Downregulation of one or both of these is thus a potential therapeutic approach for COVID-19. TMPRSS2 is a known target of the androgen receptor, a ligand-activated transcription factor; activation of the androgen receptor increases TMPRSS2 levels in various tissues, most notably the prostate. We show here that treatment with the antiandrogen enzalutamide – a well-tolerated drug widely used in advanced prostate cancer – reduces TMPRSS2 levels in human lung cells. Further, enzalutamide treatment of mice dramatically decreased Tmprss2 levels in the lung. To determine therapeutic potential, we assessed uptake of SARS-CoV-2 Spike protein pseudotyped lentivirus and live SARS-CoV-2 into human lung cells and saw a significant reduction in viral entry and infection upon treatment with the antiandrogens enzalutamide and bicalutamide. In support of this new experimental data, analysis of existing datasets shows striking co-expression of AR and TMPRSS2, including in specific lung cell types that are targeted by SARS-CoV-2. Together, the data presented provides strong evidence to support clinical trials to assess the efficacy of antiandrogens as a treatment option for COVID-19.


Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the COVID-19 pandemic, is a positive-sense single-stranded RNA coronavirus highly related to SARS-CoV, which caused the 2002 SARS pandemic 1,2 . Like SARS, COVID-19 primarily affects the respiratory system (although other organs can also be affected): symptoms are mild in some, but in others the infection can result in pneumonia, Acute Respiratory Distress Syndrome (ARDS) and death 3 . Risk factors associated with poor prognosis include age, diabetes and cardiovascular disease 4 . It has also been shown that gender is a prognostic factor, with approximately 60-70% of deaths being in men 5,6 , suggesting that sex steroid hormones may be a contributing factor to the severity of the disease. In further support of this, recent studies have shown that men with male pattern hair loss (caused by elevated androgen signalling 7 ), are at higher risk of suffering more severe COVID-19 symptoms 8, 9 . Coronaviruses have structural (Spike, Nucleocapsid, Membrane and Envelope) and non-structural (e.g. the proteases nsp3 and nsp5) proteins 10 . Cellular entry of SARS-CoV-2 requires host proteins expressed on the epithelial cell surface, most essential are Transmembrane Serine Protease 2 (TMPRSS2) and Angiotensin-Converting Enzyme 2 (ACE2) [11][12][13] . TMPRSS2 cleaves and primes the viral Spike (or S) protein; this facilitates fusion of the viral and host membranes [14][15][16][17][18] . Cellular entry is facilitated by ACE2, a terminal carboxypeptidase and type I transmembrane glycoprotein 19 . Thus, viral entry may be prevented or slowed by inhibition of ACE2 and/or TMPRSS2. TMPRSS2 is an attractive target as knockout of this protein causes no overt detrimental phenotype 20 , whereas ACE2 downregulation is associated with increased severity of SARS-induced lung injury 21 . Further, TMPRSS2 expression levels have been shown to be associated with disease severity in mouse models of coronavirus infection 22 , and its inhibition was recently shown to inhibit SARS-2-S-driven entry in lung cells 13 .
Importantly, previous studies have demonstrated that the AR is expressed in the lung 30 and studies using mice have con rmed that AR is functional in this organ 31,32 . In corroboration, in vitro studies have shown that multiple lung lines express functional AR 31,[33][34][35] . It is therefore possible that inhibition of androgen signalling, in response to antiandrogens, will reduce TMPRSS2 expression in the lung and reduce viral entry. For this reason, antiandrogens have been proposed as a treatment option for COVID-19 [36][37][38] . Here we investigate AR regulation of TMPRSS2 in the lung, and provide pre-clinical data to support the use of antiandrogens for the treatment of COVID-19.

Results And Discussion
TMPRSS2 is an androgen and antiandrogen-regulated gene ACE2 and TMPRSS2 are crucial for SARS-CoV-2 entry into cells 13 , and hence these proteins represent potential therapeutic targets for COVID-19. TMPRSS2 has been shown to be an AR target gene in prostate cancer [23][24][25] and we therefore hypothesised that the expression of TMPRSS2 could be down-regulated in response to antiandrogens. To con rm this, the AR-positive prostate cancer cell line LNCaP was seeded in hormone-depleted media for 72 h and treated with dihydrotestosterone (DHT, 10 nM) ± 10 mM of the antiandrogens bicalutamide (BIC) or enzalutamide (ENZA) for 24 h. Alterations in gene expression were quanti ed using qPCR. As expected, addition of androgen signi cantly increased TMPRSS2 expression ( Figure 1A). Importantly, the anti-androgens successfully blocked this androgen-induced up-regulation, resulting in complete inhibition of TMPRSS2 expression. A more robust induction of TMPRSS2 expression was found when using the synthetic androgen mibolerone (Supplemental Figure 1A), and this was also successfully inhibited through the addition of bicalutamide and enzalutamide, as was the TMPRSS2 expression present under full medium conditions by enzalutamide (Supplemental Figure 1B). To determine whether AR regulation of TMPRSS2 also occurs in other cell types, gene expression was investigated in the T47D breast cancer cell line (GSE62243) 39 . In agreement with the LNCaP results, TMPRSS2 was also found to be upregulated in response to DHT in this line ( Figure 1B).
TMPRSS2 and the AR are co-expressed in the lung Androgen signalling is known to be important in multiple tissues/organs. To better characterise this signalling, we previously created the AR-LUC transgenic mouse in which luciferase expression is under the control of an androgen responsive promoter, allowing for visualisation of both in vivo and ex vivo AR activity. AR signalling was found to be active in a number of tissues/organs, including the prostate, seminal vesicles, uterus and ovaries -and importantly, AR signalling was also found to be active in the lungs of male and female mice, although activity was weaker than in the reproductive organs 32 . Other studies have also demonstrated that AR signalling is active in the lung. For example, Mikkonen et al. found the AR to be predominantly expressed in type II pneumocytes and the bronchial epithelium and microarray analysis of the murine lung, and demonstrated that genes involved in oxygen transport (among other pathways) are up-regulated in murine lung tissue in response to androgen 31 .
To investigate AR and TMPRSS2 expression in different human tissues, we interrogated the Genotype Tissue Expression (GTEx) dataset 40 . We found that AR and TMPRSS2 are co-expressed in a number of tissues, and generally, TMPRSS2 is only expressed in tissues that also show detectable levels of the AR, with the exception of the pancreas (Figure 2A). Importantly, AR and TMPRSS2 were found to be coexpressed in the lung (highlighted red, also highlighted are prostate, breast (both co-expressing) and pancreas). Analysis of single cell sequencing data from lung tissue 41 , demonstrated that TMPRSS2, ACE2, AR and AR-associated pioneer factors (JUN and FOXA1) are co-expressed in Epithelial Subtype Ciliated and Alveolar Type 2 (AT2) cells ( Figure 2B). Similarly in a second single cell data set 42 , AT2 cells were among the resident lung cells with the highest expression of TMPRSS2, ACE2, and AR ( Figure 2C).
These cell types are targeted by SARS-CoV-2 43 .

TMPRSS2 expression in the lung is higher in men
In adults, men have on average 7-8 times higher levels of circulating testosterone compared to women 44 . Men are known to have more severe symptoms following SARS-CoV-2 infection (60-70% of COVID-19related deaths are in men 5, 6 ) suggesting elevated androgen signalling could be a risk factor for the disease. Further, recent studies have linked male pattern hair loss (a result of elevated androgen signalling) with more severe COVID-19 symptoms 8,9 . Since TMPRSS2 is an androgen-regulated gene, it has been proposed that elevated levels of TMPRSS2 in the lung, as a result of higher levels of androgen, might explain this gender disparity and it was therefore hypothesised that TMPRSS2 expression would be higher in male lungs compared to females. Our analysis of the GTEx dataset found no signi cant difference in AR expression levels between men and women. TMPRSS2 expression was also found to be similar between males and females ( Figure 3), but expression in the male lung was found to be slightly and signi cantly higher, in agreement with a previous study 45 . This is however, in contrast to other studies that have found no signi cant difference in TMPRSS2 expression in male and female lungs 37,46,47 . Further, Baratchian et al. found higher levels of ACE2 in the male lung and proposed that alterations in the levels of this receptor could, at least in part, explain the gender disparity in COVID-19 severity 46 . It therefore remains unclear if gender differences in TMPRSS2 expression could explain why men suffer more severe symptoms following infection with SARS-CoV-2.
TMPRSS2 expression is reduced by enzalutamide in lung cells As discussed above, the AR is expressed in human and murine lung, and shown to be active as indicated by regulation of AR-target genes in mouse lung 31 , nuclear localisation of AR in (male) human lung 31,48 , and activation of an androgen-responsive reporter gene in mouse lung 32 . TMPRSS2 expression has been previously shown to be androgen-regulated in the A549 cell line 31 . To replicate these ndings, and to expand to other lung lines, we seeded A549 (type II pneumocyte cell line; a cell type targeted by SARS-CoV-2 43 ), H1944 (lung adenocarcinoma) and BEAS-2B (bronchial epithelial) cells in hormone-depleted media (containing serum that has been charcoal-stripped to remove any traces of hormones) for 3 days then treated ± 10 nM DHT ± 10 µM enzalutamide for 24 h ( Figure 4A). In A549 and H1944, addition of DHT led to a signi cant increase in TMPRSS2 expression. This induction was blocked following treatment with enzalutamide, although this did not reach signi cance. Enzalutamide treatment in full media conditions also resulted in signi cant downregulation of TMPRSS2 in A549 (Supplementary Figure  1B). Although DHT did not induce TMPRSS2 expression in BEAS-2B, enzalutamide did appear to reduce gene expression, suggesting that the AR could be activating gene expression in a ligand independent manner in this line. A similar trend was seen across the cell lines for another known AR target gene, FKBP5; this was signi cantly up-regulated in response to DHT in A549 and H1944 and enzalutamide signi cantly reduced this induction ( Figure 4B).
To con rm AR regulation of TMPRSS2 at the protein level, A549 cells were treated as above for 48 h ( Figure 4C) before immunoblotting. DHT increased AR levels, and enzalutamide prevented this ligandmediated stabilisation of the receptor. In agreement with the qPCR data, DHT increased TMPRSS2 protein levels (approximately 2-fold) and ENZA completely blocked this induction.

TMPRSS2 expression is reduced by enzalutamide in mouse lung
To investigate the effects of enzalutamide on TMPRSS2 expression in vivo, mice were treated for three days with enzalutamide or vehicle control. Following sacri ce, lung tissue was collected and qPCR performed to quantify alterations in gene expression. While there was no signi cant change in Ar or Ace2 expression, Tmprss2 expression was signi cantly decreased after enzalutamide treatment (P<0.05, Figure  5A). Tmprss2 protein was found to be expressed in the epithelial cells and in cells of the parenchyma, as suggested by the single cell sequencing data (Figure 2), and intensity was visibly less in mice treated with enzalutamide (examples in Figure 5B). The Ar was also found to be expressed in these cell types, with nuclear localisation indicating active Ar, and enzalutamide treatment resulted in a marked decrease in Ar levels in the lung (Supplemental Figure 2). To validate these ndings, gene expression data from intact mice and mice that had been castrated (removal of testicular production of androgen) were interrogated (GSE31341) 49 . In agreement with our cell line data, castration signi cantly reduced Tmprss2 expression in the mouse lung ( Figure 5C). In the same mice, castration was also associated with an increase in Ar expression (P<0.01), expected as Ar gene transcription is downregulated in response to androgen 50 . In contrast to the results presented here, Baratchian et al. found no regulation of Tmprss2 in the mouse lung in response to enzalutamide 46 . However, in that study mice were fed enzalutamide whereas in this study oral gavage was used. The method of enzalutamide administration may therefore go some way to explain this discrepancy.
TMPRSS2 expression in lung is potentially directly regulated by nuclear receptor proteins and coregulators.
Although ChIP-Seq data for genomic AR binding in lung tissue or cells is not available, we were able to assess the cistrome of FOXA1 and JUN, known pioneer coregulatory factors for the AR 51 and other nuclear receptors. Binding of the glucocorticoid receptor (GR) was also investigated as this can bind to many of the same response elements as the AR 52 , also acetylated histone 27 (H3K27ac) as an indicator of active regulatory regions, all in A549 lung cells ( Figure 6A). Binding pro les for prostate (LNCaP) and breast (MCF-7) cell lines were included for comparison. In LNCaP cells the AR binding pattern correlates with previous ndings 24 , and con rms that AR and GR bind in the same regions, corresponding also to binding of the pioneer factor FOXA1, and these sites largely correlate with the marker of transcriptionally active regions, H3K27ac. Detailed analysis of these potential response elements by the Claessens laboratory demonstrated that an androgen response element in the enhancer region (approximately -13 kb from the transcription start site) is crucial for optimal androgen regulation of TMPRSS2 in prostate cells 24 .
The binding patterns of GR, pioneer factors and H3K27ac in lung cells, however, differ to what is seen in LNCaP cells (compare regulatory region 1 and 2). To assess if androgen response elements are present in regulatory region 2, the AR binding motif (MA0007.2, Figure 6B) from the JASPAR database, was used to detect AR target sites using methods previously described 53 . This analysis identi ed potential androgen response elements throughout the 5' region of the TMPRSS2 gene ( Figure 6A and Supplemental Table 1). Importantly, several of the potential androgen response elements were found to correlate with the GR, FOXA1, JUN, and H3K27ac peaks seen in the A549 regulatory region 2. Together, this suggests that AR (and associated factors) may directly regulate TMPRSS2 via different regulatory regions in lung and prostate. To investigate this, we performed ChIP-qPCR on these regions in LNCaP, A549 and H1944 cells ( Figure 6C). In agreement with our prediction, the AR binding sites differed between lines, with AR predominantly binding to regulatory region 1 in LNCaP and regulatory region 2 in A549. Interestingly AR binding, whilst more pronounced in regulatory region 2, was found to be present in both regulatory regions in H1944.
The DNA-binding of AR, GR, FOXA1, JUN, and H3K27ac around the TMPRSS2 gene in breast cancer cells (MCF-7) appears to be less pronounced than in prostate and lung, and the binding pattern has elements of the binding patterns in both prostate and lung cells. Importantly, AR binding in MCF-7 cells correlates with the H3K27ac, FOXA1, JUN and GR peaks located distally in the A549 regulatory region 2. Intriguingly, this region also correlates with a peak for oestrogen receptor-a (ESR1) binding in MCF-7, and oestrogen has been shown to down-regulate TMPRSS2 expression 54 . This supports the possibility of TMPRSS2 regulation by other members of the nuclear receptor superfamily, and hence further potential for pharmacological manipulation -in this case oestrogens as well as, via the GR, glucocorticoids.

Antiandrogens can successfully reduce SARS-COV-2 infection.
We have demonstrated that antiandrogens can successfully reduce TMPRSS2 expression in lung cells. To test our hypothesis that this will inhibit SARS-CoV-2 viral entry, A549 cells were treated with bicalutamide or enzalutamide for 72 h prior to transduction for 48 h with SARS-CoV-2 Spike protein pseudotyped and luciferase-expressing lentivirus ( Figure 7A). Both antiandrogens signi cantly reduced viral entry, with the latter reducing cell entry by approximately 50%. To see if antiandrogens could inhibit SARS-CoV-2 infection, TCID 50 assays were performed with the live virus ( Figure 7B). For this, A549 cells were transfected with a vector expressing ACE2 to facilitate the infection 55 , and treated with the antiandrogens for 72 h prior to infection. In agreement with the pseudotyped virus experiments, the antiandrogens signi cantly reduced SARS-CoV-2 viral titres by approximately 18-fold for enzalutamide and 13-fold for bicalutamide. The incomplete block of viral entry/infection could be a result of S-protein priming via different proteases or due to alternative, currently unknown, virus entry mechanisms. For example, TMPRSS4, has also been shown to facilitate SARS-CoV-2 cell entry in the lung 56 and therefore targeting additional proteases involved in S-priming may therefore have additive/synergistic effects.

Conclusions
The data presented here con rm a role for AR in regulation of TMPRSS2 in the lung, which may at least in part explain why men with COVID-19 have a worse prognosis compared to women. Data from prostate and breast tissue also support regulation in other organs, which may also be targeted by SARS-CoV-2. Importantly, our ndings support the hypothesis that therapies to target AR signalling could be used to transcriptionally inhibit lung TMPRSS2 expression. Further, potential regulation of TMPRSS2 by other, related receptors (revealed by cistromic analysis) opens up the possibility of additional potential opportunities for pharmacological inhibition of TMPRSS2 expression. Down-regulation of TMPRSS2 will result in attenuated spike protein priming, reducing SARS-CoV-2 interaction with ACE2 and viral entry (summarised in Figure 7C). Antiandrogens are used routinely in, or have been trialled for, the treatment of multiple diseases, including prostate cancer, breast cancer, polycystic ovarian syndrome and alopecia 57 .
They have been shown to be well tolerated in men and women [57][58][59] and therefore antiandrogen treatments should be considered as potential therapeutic, and possibly preventative, strategies for COVID-19.

Quantitative PCR analysis of gene expression in cell lines
Cells were seeded in 12 well plates and incubated + 10 nM dihydrotestosterone ± 10 mM bicalutamide or enzalutamide for 48 or 72 h. RNA was extracted using Trizol (Thermo Fisher), following the manufacturer's instructions. 250 ng of RNA was reverse transcribed using a LunaScript RT SuperMix Kit (NEB, Ipswich, MA, USA). Alterations in gene expression were quanti ed using the Luna Universal qPCR Master Mix (NEB) and a Roche 96 qPCR machine (Basel, Switzerland). TMPRSS2 data were normalised to L19 data and the 2 (-delta delta CT) method was used to calculate gene expression changes. Human qPCR primers: Analysis of TMPRSS2 expression in tissue and cell line datasets RNA-sequencing dataset v8 was downloaded from the Genotype-Tissue Expression (GTEx) Project Online Portal 63 . Gene expression was normalised (inverse normal transformation) across samples, and medians for AR and TMPRSS2 expression across each tissue was calculated. Data from RNA sequencing of isolated single nuclei, performed on surgical specimens of healthy, non-affected lung tissue from twelve lung adenocarcinoma (LADC) patients, was analysed for AR, TMPRSS2, and ACE2 expression using Eils Lab UCSC Cell browser (https://eils-lung.cells.ucsc.edu) 42 . Sequencing data from T47D cells treated with 10 nM DHT (GSE62243) 39 , and data from lungs of castrated or intact mice (GSE31341) 49 were log2 transformed. Signi cance was determined by ANOVA. Two single cell seq data sets were investigated. The rst dataset of lungs from 9 patients (GSE122960 41 ) was analysed using http://altanalyze.org/, and the second dataset of lungs from 12 donors 42 analysed using https://eilslung.cells.ucsc.edu.

Identi cation of potential AREs
The AR binding motif, MA0007.2 was obtained from the JASPAR online curated motif database 53 . Segments of DNA around the TMPRSS2 gene were scanned for potential matches for presence of the MA0007.2 AR motif using FIMO 64

ChIP-qPCR
LNCaP, A549, and H1944 cells were grown in 15 cm plates to approximately 80 % con uency, before 4 h treatment with 10nM DHT or vehicle control. Cells were xed with 4 % formalin and ChIP performed as previously published 73 . Antibodies used in overnight 4 °C immunoprecipitation were anti-AR (sc-7305, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit mouse IgG at 10 mg per sample. After DNA isolation with phenol:chloroform:isoamyl alcohol and resuspension in water, enrichment across the two potential TMPRSS2 regulatory regions was quanti ed with qPCR (primers in Supplementary Table 1).

Immunohistochemistry
Lung samples from the above mouse studies were xed in 4% formalin for 24 h, before transfer to 70% alcohol for storage before processing into wax. Sections were probed with anti-Tmprss2 antibody (ab92323, Abcam) and anti-AR (ab108341, Abcam) overnight at 4 °C at a 1/1000 dilution before detection with the Histostain-Plus IHC HRP Kit and DAB (Thermo Fisher).

SARS-CoV-2 live virus infection studies
A549 cells were seeded in 12-well plates and transiently transfected with 1ug of pCAGGS-ACE2 (synthesised by GeneArt, Thermo Fisher) using Lipofectamine 3000 reagent as described by the manufacturer (Thermo Fisher). After 24 h, cells were treated ± 10 mM bicalutamide or enzalutamide for 56-72 h. Cells were then washed with PBS and infected with SARS-CoV-2 virus. The viral strain used was SARS-CoV-2/England/IC19/202 (IC19) 75 . All work involving the use of live SARS CoV-2 virus was performed in a Containment Level 3 (CL3) laboratory (St Mary's, Imperial College London).
The virus was diluted in serum-free DMEM (supplemented with 1 % NEAA and PS) to a multiplicity of infection (MOI) of 1. The inoculum was added to A549 cells overexpressing ACE2 and incubated at 37 o C for 1 hour. The inoculum was then removed and cells maintained as described above. 24 h post infection, the culture supernatants were collected and quanti ed by TCID 50 assay on GMK Vero E6 cells as described previously 11 . Serial dilutions of the virus (in serum-free DMEM) were added in 96-well plates and cells were left for 4-5 days before they were xed with 2x crystal violet solution and analysed. TCID 50 titres were determined by the Spearman-Karber method 76 .

Statistical Analyses
Statistical analyses were performed using GraphPad PRISM (v 6.0c). For experiments with 2 treatment arms, one-tailed T-tests were performed. For analysis of more than 2 treatments, one-way ANOVA was performed with Dunnett's or Bonferroni's multiple comparison tests. For comparison of AR and TMPRSS2 levels in the human lung (GTEx dataset), Wilcoxon tests were performed. All experiments were at least 3 independent repeats, unless otherwise stated. We would like to thank Piers Aitman for discussion and image design/processing and Gilberto Serrano de Almeida for assistance with in vivo experiments. We would also like to thank the Brooke and Bevan labs and Antonio Marco for discussion of the project and Lynwen James for assistance with cell culture. Finally, we are extremely grateful to the infrastructure support provided at the CRUK Imperial College Centre, Imperial BRC and University of Essex. The GTEx project is supported by multiple funding bodies NIH, NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. Figure 3 TMPRSS2 is expressed at higher levels in the male lung compared to female lung. The GTEx dataset (40) was interrogated and expression of AR and TMPRSS2 investigated in lung tissue and dichotomized by gender, M = male (N=220), F = female (N=355). Box and whiskers, where the centre line represents the median, the box represents the 25th and 75th percentile and the bars represent the min and max values. Signi cance tested via Wilcoxon test, * P<0.05.

Figure 4
Enzalutamide successfully down-regulates TMPRSS2 in mouse lung. A) Relative expression of Ar, Tmprss2, and Ace2 mRNA from lung tissue of mice treated with enzalutamide (ENZA, n=9) or vehicle control (VC, n=8) once daily for 3 days. Expression was normalised to housekeeping genes and made relative to VC. B) Example of Tmprss2 IHC in mouse lung. Representative immunostaining of epithelial cells (*) and parenchyma/AT cells (#) in sections from 9 VC treated mice, and 8 ENZA treated mice. C) Log2 expression of Tmprss2, Ar, and Ace2 in lung tissue from male mice physically castrated (n=3) vs intact male mice (n=3) (GSE31341) (49). Statistical analysis was performed using Student T test (onetailed), * p< 0.05, ** p<0.01. TMPRSS2 is androgen regulated in lung cell lines. A549, H1944 and BEAS-2B were incubated in hormonedepleted media for 72 h and treated +/-10 nM dihydrotestosterone (DHT) +/-10 uM enzalutamide (ENZA) for 24 h. RNA was harvested, reverse transcribed and qPCR performed to quantify A) TMPRSS2 and B) FKBP5 expression. Mean of 3 independent repeats (+/-1SEM). Signi cance determined using one-way ANOVA with Dunnett's multiple comparison test, * p<0.05, *** p<0.0001. C) A549 were incubated in hormone-depleted media for 72 h and treated +/-10 nM DHT +/-10uM ENZA for 24 h. Cells were lysed and proteins separated using SDS-PAGE. Immunoblotting was performed to visualise AR and TMPRSS2 expression levels and beta-actin used as a loading control. Densitometry was performed for AR and TMPRSS2, values normalised to beta-actin and made relative to VC.

Figure 6
Potential regulatory regions in the TMPRSS2 promoter. A) ChIP sequencing peaks of AR, FOXA1, GR, and H3K27ac in LNCaP cells; FOXA1, GR, and H3K27ac in A549 lung cells; AR, H3K27ac, FOXA1, GR, ESR1 in MCF-7 breast cancer cells. The TMPRSS2 gene is highlighted in the purple shaded box and the potential regulatory region 1 is boxed in yellow, the potential regulatory region 2 is boxed in orange. Potential AREs are marked in blue boxes (MA0007.2) or green (determined by (24)) Position of potential MA0007.2 motifs around the TMPRSS2 gene separated into two regions, the rst region (regulatory region 1) covering areas of TSS and promoter/enhancers, the second region (regulatory region 2) covers more distant enhancer regions. B) AR motif MA0007.2 from JASPAR curated motif database. C) ChIP-qPCR of AR binding, and IgG control, to AR-motif containing sites within regulatory regions 1 and 2 in LNCaP, A549, and H1944 cells treated with 10nM DHT or vehicle control for 4 hours. Data is normalised to input and is an average of two independent repeats.