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01.12.2018 | Primary Research | Ausgabe 1/2018 Open Access

Cancer Cell International 1/2018

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

Zeitschrift:
Cancer Cell International > Ausgabe 1/2018
Autoren:
Sergio Regufe da Mota, Sarah Bailey, Rosemary A. Strivens, Annette L. Hayden, Leon R. Douglas, Patrick J. Duriez, M. Teresa Borrello, Hanae Benelkebir, A. Ganesan, Graham Packham, Simon J. Crabb
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s12935-018-0568-1) contains supplementary material, which is available to authorized users.

Abstract

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.
Zusatzmaterial
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 ± 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.
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