The antiandrogen enzalutamide downregulates TMPRSS2 and reduces cellular entry of SARS-CoV-2 in human lung cells

Abstract

SARS-CoV-2 attacks various organs, most destructively the lung, and cellular entry 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; androgen receptor activation increases TMPRSS2 levels in various tissues, most notably 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 and in mouse lung. Importantly, antiandrogens significantly reduced SARS-CoV-2 entry and infection in lung cells. In support of this experimental data, analysis of existing datasets shows striking co-expression of AR and TMPRSS2, including in specific lung cell types 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.

Fig. 1. TMPRSS2 is an androgen-regulated gene in cells from different tissues.

a LNCaP cells were incubated in hormone-depleted media for 72 h and treated with ±10 nM dihydrotestosterone (DHT) ± 10 μM bicalutamide (BIC) or enzalutamide (ENZA) for 6 h. RNA was harvested, reverse transcribed and qPCR performed to quantify TMPRSS2 expression. Mean of three independent repeats (±1 SEM). One-way ANOVA with Tukey’s multiple comparison test **** Vehicle Control (VC) v DHT – p = 8.6 × 10^–11, DHT v BIC – p = 4.9 × 10^–11, DHT v DHT + BIC – p = 7.3 × 10^–10, DHT v ENZA – p = 4.0 × 10^–11, DHT v DHT + ENZA – p = 1.0 × 10^–10. b Data from T47D cells (GSE62243, N = 3) treated with 10 nM DHT were analysed for TMPRRS2 expression. One-tailed t-test, *p = 0.0302. Source data are provided as a Source Data file.

Fig. 2. TMPRSS2 and the AR are expressed in the lung.

a The GTEx dataset was interrogated and median expression of AR was plotted against median TMPRSS2 expression. Data are expressed as each gene normalised across all tissues (z-score). Highlighted are prostate (n = 245), lung (n = 578), pancreas (n = 328) and breast (n = 459). b Single-cell analysis of lung tissue with each bar representing AR, TMPRSS2, ACE2, FOXA1, JUN expression within individual cells of each specified cell type. c Single-cell analysis of lung tissue was interrogated and a breakdown is shown of AR, TMPRSS2 and ACE2 expression levels in specific lung cell types (X-axis); Y-axis denotes gene mRNA CPM.

Fig. 3. TMPRSS2 is expressed at higher levels in the male lung compared to female lung.

The GTEx dataset was interrogated and expression of AR and TMPRSS2 investigated in lung tissue and dichotomised by gender, M = male (N = 220), F = female (N = 355). Box and whiskers: the centre line represents the median, the box represents the 25th and 75th percentile and the bars represent the min and max values. One-tailed t-test, *p = 0.0403.

Fig. 4. TMPRSS2 is androgen regulated in lung cell lines.

A549, H1944 and BEAS-2B were incubated in hormone-depleted media for 72 h and treated with ±10 nM dihydrotestosterone (DHT) ± 10 μM enzalutamide (ENZA) for 24 h. RNA was harvested, reverse transcribed and qPCR performed to quantify a TMPRSS2 and b FKBP5 expression. Mean of at least three independent repeats (±1 SEM). Significance determined using one-way ANOVA with Dunnett’s multiple comparison test. TMPRSS2: A549 Vehicle Control (VC) v DHT p = 0.0190, DHT v ENZA p = 0.0228; H1944 VC v DHT p = 0.0410, DHT v ENZA p = 0.0497. FKBP5: A549 VC v DHT p = 0.0250, DHT v ENZA p = 0.0493, DHT v DHT + ENZA p = 0.0285; H1944 VC v DHT p = 5.7 × 10^–6, DHT v ENZA p = 7.1 × 10^–5, DHT v DHT + ENZA p = 1.7 × 10^–5. c A549 were incubated in hormone-depleted media for 72 h and treated with ±10 nM DHT ± 10 μM ENZA for 24 h. Cells were lysed and proteins separated using SDS-PAGE. Immunoblotting was performed to visualise AR and TMPRSS2 expression levels and β−actin used as a loading control. Densitometry was performed for AR and TMPRSS2, values normalised to β−actin and made relative to VC. Representative immunoblots blots – A549 n = 3, H1944 n = 2. Source data are provided as a Source Data file.

Fig. 5. Enzalutamide downregulates 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. Mean ± 1 SE. Student’s t-test (one-tailed) *p = 0.0367. b Example of Tmprss2 IHC in mouse lung. Representative immunostaining of epithelial cells (*) and parenchyma/AT cells (#) in sections from n = 9 VC-treated mice, and n = 8 ENZA-treated mice. Bar = 50 μm 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). Mean ± 1 SE. Student’s t-test (one-tailed), **p = 0.0018, *p = 0.0081. Source data are provided as a Source Data file.

Fig. 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). Position of potential MA0007.2 motifs around the TMPRSS2 gene separated into two regions, the first 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 10 nM dihydrotestosterone (DHT) or vehicle control (VC) for 4 h. Data are normalised to input and made relative to AR-IP VC. Data are the mean of three independent repeats. A maximum of one outlier was removed from each treatment using Grubbs’ test (alpha = 0.05). Source data are provided as a Source Data file.

Fig. 7. Antiandrogens reduce SARS-CoV-2 entry and infection in lung cells.

a A549 cells were seeded in full media and treated with ±10 μM bicalutamide (BIC) or enzalutamide (ENZA) for 72 h prior to transduction with SARS-CoV-2 Spike protein or VSV-G control pseudotyped lentiviral particles expressing luciferase. Cells were left for an additional 48 h and luciferase assays performed. Luciferase data were normalised to total protein content. Mean of three independent repeats ± 1 SE. One-way ANOVA with Sidak’s multiple comparison test. Vehicle Control (VC) v BIC p = 1.3 × 10⁻⁴, VC v ENZA p = 2.1 × 10⁻⁵. b A549 were seeded in full media, transfected with ACE2 and treated with ±10 μM BIC or ENZA for 72 h prior to infection with SARS-CoV-2. Infectious titres of SARS-CoV-2 in supernatants of A549-ACE2 were determined with TCID₅₀ assays after 24 h of infection. n = 4, mean of two independent experiments repeated in duplicate ± 1 SE. One-way ANOVA with Sidak’s multiple comparison test. VC v BIC p = 2.0 × 10⁻⁶, VC v ENZA p = 2.5 × 10⁻⁶. c Schematic representation of how targeting the AR could reduce SARS-CoV-2 entry. Source data are provided as a Source Data file.