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Figure 7. Model describing how exposure to androgens regulates splicing patterns in prostate cancer cells. ; Androgen exposure leads to transcription of the gene encoding the master splicing regulator protein ESRP2. This promotes epithelial splicing patterns within prostate cancer cells. Lower levels of circulating androgens after ADT lead to transcriptional repression of ESRP2. This results in a dampening of epithelial splicing patterns, and production of normally mesenchymal splice patterns including for the FLNB, CTNND1 and MAP3K7 genes. This image was created using BioRender.

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Figure 4. An androgen steroid hormone-ESRP2 axis controls alternative splicing in prostate cancer cells. ; (A) ESRP2-regulated exons are frequently also controlled by androgens in prostate cancer cells. 31/48 of the ESRP target exons (identified by RNAseq analysis of LNCaP cells depleted of ESRP1 and ESRP2) were regulated in the opposite direction in LNCaP cells treated by androgens (10nM R1881) for 48 hr (which would induce ESRP2 protein expression). Plotting the splicing responses to androgen stimulation with those after ESRP1/ESRP2 depletion revealed a negative correlation (slope = −0.66+/−0.09, Rsquare = 0.64, p

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Figure 6. Pharmacological inhibition of AR function switches ESRP2-dependent splicing patterns. ; (A) ESRP2 mRNA expression in cells grown in steroid deplete (SD) conditions, and after addition of androgens (A+) (quantified by real-time PCR from three biological replicates). Androgen-mediated activation of ESRP2 expression was inhibited in the presence of 10 μM of the anti-androgen bicalutamide (Casodex). Cells were cultured for 24 hr. The p value shows the statistical significance that was calculated using a t test between the A + vehicle and the A + casodex samples. (B–C) Capillary gel electrophoretogram showing RT-PCR analysis the splicing response + /- 24 hr Casodex treatment for exons that are normally (B) activated or (C) repressed by androgens (three biological samples shown, statistical significances were calculated using a t test, with the exception of CTNND1 where there was zero inclusion detected for exons 2 and 3 before depletion of ESRP2, and FN1 where there was zero inclusion of exon 25 before Casodex treatment).

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Figure 1. ESRP2 is a direct target for AR regulation in prostate cancer cells. ; (A) Analysis of RNAseq data from human prostate cancer pre- and post- androgen deprivation therapy (ADT) (Chen et al., 2018; Rajan et al., 2014) shows that there is a significant downregulation of ESRP1 and ESRP2 mRNA following ADT in all seven patients tested (p=6e-04, Mann Whitney U test). (B–C) RNAseq data from LTL331 patient-derived xenografts grown in mice (Akamatsu et al., 2015) show a greater reduction in (B) ESRP2 mRNA levels following castration compared to (C) ESRP1 mRNA levels. (D) Western blot analysis of ESRP2 levels in a range of prostate cancer cell lines (actin was used as a loading control). (E) Western blot analysis of ESRP1 levels in prostate cancer cell lines. (F) Real-time PCR analysis of ESRP2 and ESRP1 mRNAs in LNCaP cells grown in steroid deplete (SD) or androgen (A+) treated conditions for 24 hr (statistical significance calculated by t test). (G) Real-time PCR analysis of ESRP2 mRNA in RWPE-1 cells grown in steroid deplete (SD) or androgen (A+) treated conditions for 24 hr. (H) Western blots analysis of ESRP1 and 2 protein in LNCaP cells treated with 10nm R1881 (androgens) for 24 and 48 hr. (I) Quantitative analysis (real-time PCR) of ESRP2 mRNA expression over a 24 hr time course following androgen exposure. (J) Real-time PCR analysis of AR-ChIP performed in LNCaP cells treated with 10nM R1881 for 24 hr revealed AR binding proximal to the ESRP2 gene. (K) Induction of ESRP2 is evident in LNCaP cells treated with R1881 concentrations between 1 to 100 nM (p value of 0.029 is for the comparison between 0 nm and 10nm R1881). Statistical significances were calculated by t tests, apart from (A) which used a Mann Whitney U test, and H which used Two-way ANOVA. Real time PCR analyses used at least three independent biological replicates (RNA prepared from separate samples), apart from the AR ChIP (panel I) for which each value shown is a mean of 3 technical replicates.

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Figure 6—figure supplement 1. siRNA depletion of the AR switches splicing of ESRP2- regulated exons. ; (A) Western blot showing levels of the AR, ESRP2 and actin in samples of LNCaP cells following growth in steroid deplete media (SD), or plus androgens for 48 hr (A+). Cells were transfected with esiRNAs designed to deplete the AR, or a control siRNA (NTesiRNA). Statistical significance was calculated using a t test. (B) Capillary gel electrophoretograms showing RT-PCR analysis of 3 biological replicate RNA samples, measuring splicing inclusion levels for FLNB exon 30 and TUFT1 exon two after siRNA depletion of the AR or treatment with a control siRNA.

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Figure 3—figure supplement 1. Synthetic pathway for C19 and C18 steroids and the results of cross-reactivity/interference testing for EIA. ; (A) Synthetic pathway for C19 and C18 steroids. The gray boxes indicate steroids that we tested for cross-reactivity/interference, and blue steroids are those tested by the manufacturer. Estradiol is shown in red. (B) Table listing results of all our cross-reactivity/interference tests for glucocorticoids, androgens, estrogens and other neuroactive steroids. These results showed that the EIA used has little to no cross-reactivity with non-estrogens.

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Castration delays epigenetic aging and feminizes DNA methylation at androgen-regulated loci

• Authors : Sugrue, Victoria J; Zoller, Joseph Alan; Narayan, Pritika ...

• Source: eLife ; volume 10 ; ISSN 2050-084X

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Androgen-regulated transcription of ESRP2 drives alternative splicing patterns in prostate cancer

• Authors : Munkley, Jennifer; Li, Ling; Krishnan, S R Gokul ...

• Source: eLife ; volume 8 ; ISSN 2050-084X

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