TMPRSS2 is an androgen and antiandrogen-regulated gene
ACE2 and TMPRSS2 are crucial for SARS-CoV-2 entry into cells [11], and hence these receptors represent potential therapeutic targets for COVID-19. TMPRSS2 has been shown to be an AR target gene in prostate cancer (e.g. [19-21]) and we therefore hypothesised that the expression of the gene could be down-regulated in response to antiandrogens. To confirm this, the AR-positive prostate cancer cell line LNCaP was seeded in hormone-depleted media for 72 hrs and treated with the synthetic androgen mibolerone (MIB, 1 nM) and/or the antiandrogen enzalutamide (ENZA, 10 mM) for 24 hrs. Alterations in gene expression were quantified using qPCR. As expected, the addition of androgen significantly increased TMPRSS2 expression (approximately 35-fold, Figure 1A). Importantly, enzalutamide successfully blocked this androgen-induced up-regulation, resulting in an almost complete inhibition of TMPRSS2 expression. To determine whether AR regulation of TMPRSS2 also occurs in other cell types, gene expression was investigated in two breast cancer cell lines, MCF-7 (GSE99626) and T47D (GSE62243) [39]. In agreement with the LNCaP results, TMPRSS2 was also found to be upregulated in response to androgen in the breast lines, albeit weakly (Figure 1B and C).
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 [27]. Other studies have also demonstrated that AR signalling is active in the lung. For example, Mikonnnen 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 in response to androgen [26].
To investigate AR and TMPRSS2 expression in different human tissues, we interrogated the Genotype Tissue Expression (GTEx) dataset [52]. 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 co-expressed in the lung (highlighted red, also highlighted are prostate, breast (both co-expressing) and pancreas). Analysis of single cell sequencing data from lung tissue [38], demonstrated that the AR is expressed in most cell types, with highest expression in club (bronchiolar exocrine cells), alveolar type 2 (AT2), fibroblast and endothelial cells. TMPRSS2 was found to have a fairly uniform expression across cell types, but was most highly expressed in the AT1 and AT2 luminal cells (Figure 2B,C). Significantly, these are the cell types targeted by SARS-CoV-2 [53]. AT1 and AT2 cells also demonstrated highest ACE2 expression, and measurable AR expression.
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 [54]. It was therefore hypothesised that TMPRSS2 expression would be higher in male lungs compared to females. Analysis of the GTEx dataset confirmed this, with TMPRSS2 expression significantly higher in the male lung (Figure 3). Interestingly, there was no significant difference in AR expression levels between men and women, suggesting the higher levels of TMPRSS2 expression are a result of increased AR activity due to circulating androgen levels, rather than higher AR levels. This would support the theory that the observed worse prognosis in men following SARS-CoV-2 infection (60-70% of COVID-19-related deaths are in men [5, 6]) is at least in part due to elevated expression of TMPRSS2 as a consequence of higher levels of androgen. In light of the recent studies linking male pattern hair loss with more severe COVID-19 symptoms [8, 9], it would be of interest to compare TMPRSS2 expression levels in men with and without this androgen-associated form of hair loss.
TMPRSS2 expression is reduced by enzalutamide in A549 cells
As discussed above, the AR is expressed in human and murine lung, and has been shown to be active. To investigate AR regulation of TMPRSS2 in the lung, we used the A549 human type II pneumocyte cell line; a cell type targeted by SARS-CoV-2 [53]. Immunoblotting confirmed AR expression: in agreement with the GTEx data, the AR is expressed in the lung cell line and levels are approximately 10-fold lower than in the prostate LNCaP line (Figure 4A).
TMPRSS2 expression has been previously shown to be androgen-regulated in the A549 cell line [18]. To replicate these findings, we seeded A549 cells in hormone-depleted media (containing serum that has been charcoal-stripped to remove any traces of hormones) for 3 days and treated with the synthetic androgen MIB. However, under these conditions TMPRSS2 was undetectable by qPCR (data not shown). The experiment was therefore repeated for A549, and LNCaP, in media supplemented with 10% full serum, with/without enzalutamide. Fetal calf serum has been shown to contain castrate levels of testosterone, which LNCaP cells metabolise to produce physiologically relevant intracellular levels of dihydrotestosterone, sufficient to promote their growth [55]. Since this might not be the case in A549 cells, a final concentration of 10nM MIB was also added to both cell lines throughout the experiment. In these conditions, TMPRSS2 was expressed at detectable levels and, importantly, enzalutamide potently down-regulated TMPRSS2 expression in both LNCaP and A549 after 48 and (more so) 72 hours (Figure 4B). This therefore confirms that antiandrogens could be used to down-regulate TMPRSS2 expression in lung cells.
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 sacrifice, lung tissue was collected and qPCR performed to quantify alterations in gene expression. While there was no significant change in Ar or Ace2 expression, Tmprss2 expression was significantly decreased after enzalutamide treatment (P<0.05, Figure 5A). To validate these findings, expression data from intact mice and mice that had been castrated (removal of testicular production of androgen) were interrogated (GSE31341) [40]. In agreement with our cell line data, castration significantly reduced Tmprss2 expression in the mouse lung (Figure 5B). 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 [56].
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 [57] 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 [58], also acetylated histone 27 (H3K27ac) as an indicator of active regulatory regions, all in A549 lung cells (Figure 6A). Binding profiles for prostate (LNCaP) and breast (MCF-7) cell lines were included for comparison. In LNCaP cells the AR binding pattern correlates with previous findings [20], and confirms 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 lab 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 [20].
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 [31]. This analysis identified potential androgen response elements throughout the 5’ region of the TMPRSS2 gene (Figure 6A and 6C). 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.
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. This therefore provides further evidence that this region contains a functional androgen response element(s). Intriguingly, this region also correlates with a peak for oestrogen receptor-a (ESR1) binding in MCF-7. This supports the possibility of TMPRSS2 regulation by other members of the nuclear receptor superfamily, and hence further potential for pharmacological manipulation by their ligands – in this case oestrogens/antioestrogens as well as, via the GR, glucocorticoids, DHT/antiandrogens via the AR.