LSD1 inhibition attenuates androgen receptor V7 splice variant activation in castration resistant prostate cancer models

Background: Castrate resistant prostate cancer (CRPC) is often driven by constitutively active forms of the androgen receptor such as the V7 splice variant (AR-V7) and commonly becomes resistant to established hormonal therapy strategies such as enzalutamide as a result. The lysine demethylase LSD1 is a co-activator of the wild type androgen receptor and a potential therapeutic target in hormone sensitive prostate cancer. We evaluated whether LSD1 could also be therapeutically targeted in CRPC models driven by AR-V7. Methods: We utilised cell line models of castrate resistant prostate cancer through over expression of AR-V7 to test the impact of chemical LSD1 inhibition on AR activation. We validated findings through depletion of LSD1 expression and in prostate cancer cell lines that express AR-V7. Results: Chemical inhibition of LSD1 resulted in reduced activation of the androgen receptor through both the wild type and its AR-V7 splice variant forms. This was confirmed and validated in luciferase reporter assays, in LNCaP and 22Rv1 prostate cancer cell lines and in LSD1 depletion experiments. Conclusion: LSD1 contributes to activation of both the wild type and V7 splice variant forms of the androgen receptor and can be therapeutically targeted in models of CRPC. Further development of this approach is warranted

Abstract 3584: lysine specific demethylase 1 inhibition attenuates enzalutamide resistant androgen receptor V7 splice variant activation

Prostate cancer remains one of the leading causes of cancer death in men worldwide. The majority of prostate cancer is initially hormone dependent highlighting the central role of the androgen receptor (AR) pathway in this disease. However, in a significant number of the cases, patients relapse and develop castration resistant prostate cancer (CRPC) through a variety of molecular mechanisms, many of which include AR modifications. One common resistant mechanism is through an AR-V7 splice variant lacking the C-terminal ligand-binding domain (LBD) but retaining the transactivating N-terminal domain. AR-V7 is constitutively active. Emerging data imply that hormonal therapy with the AR antagonist enzalutamide, which is highly effective in some CRPC patients, becomes ineffective in the face of AR-V7 consistent with the loss of its LBD binding site.Lysine-specific demethylase 1 (LSD1) is a co-activator of the AR pathway which interacts with the AR to promote androgen-dependent transcription of target genes by ligand-induced demethylation of mono- and dimethylated histone 3 at Lys 9. In this work we identified LSD1 as new potential target for treatment of CRPC resistant to enzalutamide treatment through AR-V7 expression.We used reporter assay experiments in human embryonic kidney 293 cells, deficient for androgen receptor, to investigate the transactivation control of the putative androgen receptor responsive element (ARE) in the presence of the wild type (WT) form and the constitutively active V7 variant of the AR. Using this approach we show that enzalutamide effectively inhibits the activation of the ARE by the AR-WT but not the AR-V7 variant upon stimulation with dihydrotestosterone (DHT). However, treatment with a LSD1 inhibitor shows reduced transcription of the ARE promoter with the AR-wt with DHT stimulation and AR-V7 with and without DHT stimulation.Treatment of an androgen-sensitive human prostate adenocarcinoma cell line (LNCaP) with DHT shows an increase in the expression of prostate specific antigen (PSA) gene, an AR transcriptional target. However when treated with LSD1 inhibitors this activation was impaired or completely blocked as PSA levels were similar to those in non DHT treated sample. Moreover we show that treatment of LNCaP cells with LSD1 inhibitors trigger apoptosis as indicated by PARP cleavage.Together this results show that by targeting LSD1, a co-activator of the AR, we can inhibit the expression of AR target genes in a clinically relevant model of enzalutamide resistant AR-V7 expressing CRPC. LSD1 holds potential as a target for cancer therapy in castration resistant prostate cancer.This work was supported by Prostate Cancer UK

MOESM1 of LSD1 inhibition attenuates androgen receptor V7 splice variant activation in castration resistant prostate cancer models

Additional file 1: FIgure S1. Figure S1 Molecular structure of the LSD1 inhibitors used in this work. Figure S2. In vitro LSD1 inhibition assay for the indicated compounds. Data points show percentage of LSD1 activity when compared with the DMSO treated sample for three separate experiments. Figure S3. Cell proliferation assays for the indicated compounds in LnCAP prostate cancer cells. Data points show percentage luminescence compared to DMSO solvent exposed control samples after 72 h for a minimum of three separate experiments in each case. Figure S4. Androgen response element (ARE) luciferase reporter assay, in response to androgen receptor (AR) antagonists, for the AR wild type (WT) or V7 splice variant forms. (A) Luciferase reporter assay of ARE promotor activation in HEK293 cells co-transfected, as indicated, with either AR WT or V7 expression vectors, and incubated as indicated with dihydrotestosterone (DHT; 1nM), enzalutamide (10uM) or apalutamide (10uM). Data are mean values Âą standard deviation from four replicate determinations. P values were derived using a two-way ANOVA for comparison followed by Tukey's multiple comparison post hoc tests. (B) Western blot to confirm AR expression following transfection of either the WT or V7 or Q640X splice variant forms or an untransfected sample, as indicated, in HEK293 cells. ****, P < 0.0001; ns, non-significant; NT, non-transfected. Figure S5. Western blot analysis of LSD1 expression for different clones obtained after the CRISPR experiment. (A) immunoblot for the N-terminus of human LSD1. (B) immunoblot for the C-terminus of human LSD1. Actin expression is shown as a protein loading control