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 5

TRA2B binding to CE3 is required for AR-V7 synthesis.

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TRA2B binding to CE3 is required for AR-V7 synthesis. (A) Diagrammatic r...
(A) Diagrammatic representation of TRA2B binding sites (AGAA) and the upstream exonic splicing enhancer (ESE) sequence in mRNA encompassing AR-V7-encoding CE3. (B) CWR22Rv1 cells were subject to RNA immunoprecipitation using either anti-TRA2B or control antibodies prior to qRT-PCR to quantify percentage input of TRA2B interaction with AR-V7 and control RPL13A transcripts (*P < 0.05, **P < 0.01, and ***P < 0.001, as calculated using a 1-way ANOVA from at least 3 independent experiments). (C) Diagrammatic representation of TRA2B binding sites encompassing AR-V7-encoding CE3 and the phosphorodiamidate morpholino oligomers (PMOs) targeting a region encompassing the ESE of the AR pre-mRNA transcript (red dotted line). (D) 10× original magnification live-cell imaging of CWR22Rv1 cells transfected with and without 10 μM fluorescein-labeled CE3-targeting PMO for 48 hours to validate the transfection pipeline. (E) Cells transfected as in D with control or CE3-targeting PMOs were subject to RNA immunoprecipitation using either anti-TRA2B or control antibodies before RT-qPCR analysis to assess AR-V7 transcript enrichment by TRA2B in the presence and absence of control or CE3-targeting PMOs (**P < 0.01 as calculated from 3 independent replicates using a 1-way ANOVA). CWR22Rv1 (F) and VCaP (G) were transfected with 10 μM CE3-targeting or control PMO for 48 hours prior to RT-qPCR analysis expression of the indicated mRNAs. qPCR data comprise n = 3 independent biological replicates, plotted as mean ± SEM and subject to a 1-way ANOVA (***P < 0.001).