Splicing factor TRA2B enhances synthesis of androgen receptor variant AR-V7 in prostate cancer cells
Nicholas Brittain, Alec Paschalis, Ryan Nelson, Beth Adamson, Laura Walker, Ruaridh Duncan, Graham R. Smith, Suzanne McGill, Richard J.S. Burchmore, Denisa Bogdan, Juan M. Jiménez-Vacas, Jonathan Welti, Wei Yuan, Craig N. Robson, Pasquale Rescigno, Sara Luzzi, Adam Sharp, Johann de Bono, Luke Gaughan
Nicholas Brittain, Alec Paschalis, Ryan Nelson, Beth Adamson, Laura Walker, Ruaridh Duncan, Graham R. Smith, Suzanne McGill, Richard J.S. Burchmore, Denisa Bogdan, Juan M. Jiménez-Vacas, Jonathan Welti, Wei Yuan, Craig N. Robson, Pasquale Rescigno, Sara Luzzi, Adam Sharp, Johann de Bono, Luke Gaughan
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Research Article Clinical Research Oncology

Splicing factor TRA2B enhances synthesis of androgen receptor variant AR-V7 in prostate cancer cells

Abstract

Treatment of locally advanced and metastatic prostate cancer (PC) with androgen receptor–targeting (AR-targeting) therapies has limited durability, with disease eventually progressing to castrate-resistant PC (CRPC). Constitutively active AR splice variants (AR-Vs), such as AR-V7, play a key role in driving treatment resistance and disease progression. Importantly, the failure to attenuate AR-V function represents a major unmet clinical need, and as such, defining how AR-Vs are generated is likely to yield new therapeutic targets. Our knowledge of factors that mediate splicing of AR-V–encoding mRNAs remains limited. Here, we have employed an RNA-targeting CasRx approach to identify selective protein interactors of AR-V7 mRNA in PC. TRA2B and its ortholog, TRA2A, were identified as splicing regulators of AR transcripts that facilitate AR-V synthesis at the expense of full-length AR isoforms. TRA2B expression correlated with AR-V7 transcript in CRPC and attenuation of TRA2-mediated splicing diminished PC cell growth. Exploiting TRA2B function may therefore provide new therapeutic opportunities in advanced disease.

Authors

Nicholas Brittain, Alec Paschalis, Ryan Nelson, Beth Adamson, Laura Walker, Ruaridh Duncan, Graham R. Smith, Suzanne McGill, Richard J.S. Burchmore, Denisa Bogdan, Juan M. Jiménez-Vacas, Jonathan Welti, Wei Yuan, Craig N. Robson, Pasquale Rescigno, Sara Luzzi, Adam Sharp, Johann de Bono, Luke Gaughan

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Figure 1

Validating selective dCasRX binding to AR-V7 mRNA.

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Validating selective dCasRX binding to AR-V7 mRNA. (A) Diagrammatic repr...
(A) Diagrammatic representation of the strategy to detect splicing regulators of pathogenic AR-Vs. When positioned at CE3 with a relevant gRNA, dCasRx-APEX2 can biotinylate proximal proteins for subsequent enrichment with magnetic streptavidin beads and protein identification by mass spectrometry. Stable doxycycline-inducible CWR22Rv1 derivatives expressing HA-CasRx (CWR22Rv1-iCasRX), an HA-dCasRx-APEX2 fusion (CWR22Rv1-idCasRX-APEX2), or parental control were treated with 1 μg/ml doxycycline for 72 hours prior to (B) Western analysis using anti-HA, -AR, and -α-tubulin antibodies or (C) RT-qPCR to assess relative abundance of FL-AR and AR-V7 transcripts. RT-qPCR data comprise n = 3 independent biological replicates, plotted as mean ± SEM. (D) Diagrammatic representation of the AR gene with position of AR CE3-targeting AR guide RNA (gAR) indicated. (E) CWR22Rv1-iCasRX were transfected with 25 nM of either a nontargeting gRNA (gNT) or gAR and induced with 1 μg/ml doxycycline for 72 hours before RNA was extracted for RT-qPCR and levels of FL-AR and AR-V7, -V1, -V6, and –V9 mRNAs were assessed. A 2-tailed unpaired t test was used for determination of statistical significance (*P < 0.05). (F) The experimental setup in E was repeated, and protein levels of AR-FL, AR-Vs, and AR-V7 were analyzed. α-tubulin was used as a loading control. Western blot is representative of n = 3 independent biological replicates. (G) CWR22Rv1-idCasRx-APEX2 were transfected with either gNT or gAR gRNAs and induced with doxycycline for 72 hours before incubation with biotin analine (BAn) and H2O2 for 2 hours and 2 minutes, respectively. Total RNA was extracted followed by streptavidin enrichment of total RNA extracts. A negative control induced with doxycycline but untreated with BAn/H2O2 was also included. RNA dot blot was performed using streptavidin-HRP. Methylene blue stain was used to confirm presence of RNA in each sample. (H) CWR22Rv1-idCasRx-APEX2 were transfected with either gNT or gAR gRNAs and induced with doxycycline for 72 hours before incubation with biotin analine (BAn) and H2O2 for 2 hours and 2 minutes, respectively. Total RNA was extracted and an RNA pulldown assay was performed with streptavidin prior to RT-qPCR to quantify levels of prespliced and postspliced CE3 mRNA enrichment between gAR and gNT-transfected samples. Data comprise n = 3 independent biological replicates, plotted as mean ± SEM. A 2-tailed unpaired t test was used for determination of statistical significance (*P < 0.05). (I) Western blot depicting levels of AR-FL and AR-V protein in CW22Rv1-idCasRx-APEX2 induced and transfected with the same gRNAs as in H. α-tubulin was used as a loading control.