1. Evaluation Summary:

    Truncated splice variants of the androgen receptor (AR) lacking a ligand-binding domain are thought to contribute to therapeutic resistance to antiandrogens in advanced prostate cancer. In this manuscript, the authors show that AR-V7, the most well-studied such truncated variant, displays a different mechanism of nuclear targeting and interaction with chromatin compared to the full-length AR. This work provides new insights into how AR-V7 may contribute to the pathology of Castrate-Resistant Prostate Cancer and will be of interest to researchers trying to improve prostate cancer therapies.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    In this study, Kim et al. perform comparative studies on the nuclear import dynamics of AR-FL versus a truncated, ligand-independent splice variant, AR-V7. Their main findings are as follows:

    1. Under basal conditions without ligand, AR-FL remains largely in the cytoplasm, as does another splice variant of the AR, AR-v567. In contrast, AR-V7 exhibits fast nuclear import kinetics and 75% of AR-V7 accumulates in the nucleus by 90 minutes after microinjection.

    2. In contrast to AR-FL, which is imported into the nucleus through a typical importin a/b import mechanism, AR-V7 is not dependent on either importin-b or microtubules for nuclear import.

    3. When AR-V7 and AR-FL are co-expressed in the same cell, the nuclear localization of AR-FL increases two-fold over AR-FL expressed alone.

    4. AR-V7 is transcriptionally active in the nucleus but exhibits very short chromatin residence time as assayed by fluorescence recovery after photobleaching as well as high intranuclear mobility.

    AR-V7 was proposed as a biomarker of resistance to antiandrogen therapies several years ago but the functional significance of AR-V7 and other AR splice variants in driving resistance has remained unresolved, as have the precise mechanisms by which these variants contribute to AR signaling relative to AR-FL. The main strength of this manuscript is that it performs careful characterization of one aspect of AR biology - namely the nuclear import kinetics - in AR-Fl, AR-V7 and AR-v567. The data clearly show that AR-V7 exhibits distinct nuclear import kinetics from AR-FL in the experimental systems used. There remain some unanswered questions - namely: 1) what is the precise nuclear import machinery used by AR-V7? And 2) is the mechanism of AR/AR-V7 import in physiologic contexts similar to that observed in the ectopic expression systems used in this study?

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  3. Reviewer #2 (Public Review):

    The data presented in this manuscript are both novel and seminal in our understanding of the molecular pathways for ARv7 driven progression of castration resistant prostate cancer. This data documents an entirely novel mechanism for this variant's nuclear uptake and transcriptional targeting. This novel information will be critical in developing therapeutic approaches to selectively inhibit ARv7 oncogenic abilities. As sole critique, it would be very helpful if the authors could include a discussion of how this new understanding of the molecular pathways of ARv7 can be used to develop new approaches for castration resistant prostate cancer.

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  4. Reviewer #3 (Public Review):

    The emergence of AR-V7 and other AR splice variants in patient tumors has been shown to track with inferior response to AR targeted and chemotherapies in prostate cancer. Using a series of sophisticated imaging techniques the authors have determined that the AR-V7 uses a mechanism for nuclear import distinct from the FL-AR and the AR-V567 variant. AR-V7 also appears to have very fast intranuclear mobility, a characteristic shown to be associated with antagonist bound AR and yet AR-V7 efficiently activates transcription of a reporter gene and at least a subset of AR target genes. This study provides new insights into how AR-V7 may contribute to the pathology of CRPC.
    Although it is well accepted that the AR-Vs serves as a strong biomarker for resistance to antiandrogen treatment, it is still being debated as to whether and how AR-Vs contribute to the pathology of castration resistance prostate cancer. In this regard, significant efforts have been invested to understand how these splice variants work and how to best target them. Several models of have been proposed, some of which suggested that the AR-Vs can dimerize with the full-length (FL) receptor and require FL-AR for activity, others indicated that AR-V has unique activities independent of FL-AR. Using a series of sophisticated imaging techniques, the authors have addressed some of these unresolved issues in the field, in particular determining that AR-V7 uses a mechanism for nuclear import distinct from the FL-AR and the AR-V567 variant. AR-V7 also appears to have very fast intranuclear mobility, a characteristic shown to be associated with antagonist bound AR; and yet, AR-V7 efficiently activates transcription of a reporter gene and at least a subset of AR target genes. Overall, this is a valuable study, and the authors are to be commended for the high-quality figures and illustrations. Specific strengths include the following points:

    1. It has been observed that the AR-V7 predominantly resides in the nucleus; however, the mechanism(s) by which AR-V7 used for nuclear import is not clear. Using live imaging combined with pharmacological and genetic approaches the authors have determined that the importin / complex is required for FL-AR, but not AR-V7 nuclear import. However, nucleoporin complex and Ran-GTP activity are required for FL-AR import, as expected, and are at least partially required for AR-V7 nuclear accumulation. These series of studies have confirmed previously finding that AR-V7 uses a mechanism for nuclear import, distinct from the FL-AR and the AR-V567 variant.

    2. Using mutagenesis of the D-box and DNA binding mutants, the authors have also contrasted the structural requirements within FL-AR vs AR-V7 for nuclear localization and transcription. It was determined that the dimerization surface is required for AR-V7 nuclear retention but is dispensable for FL-AR. However, disruption of DNA binding with a single point mutation has demonstrated that DNA binding is not an obligatory step for FL-AR and AR-V7 nulcear retention. This series of experiments have enhanced our understanding of the nuclear cytoplasmic dynamics of AR-V7 and how it differs from FL-AR and suggests that interfering with the dimerization interface may be a means by which AR-V7 can be targeted.

    3. Using FRAP and photoconvertible fluorescent protein tag, the authors were able to track the intranuclear dynamics of AR and AR-V7 in real time, which is a strength of this study. They have determined that AR-V7 has higher sub-nuclear mobility compared to FL-AR and that DNA binding is required for even the short residence time of this mutant AR on the chromatin. Together these data suggested that AR-V7 and FL-AR may use different means to activate transcription.

    There are some weaknesses that could be addressed to improve the work:

    1. Most of the studies were done in PC3 AR negative cell line. It would be helpful to confirm some the key findings in AR positive cell line as the import mechanism may not be the same in AR negative vs AR-positive cell lines.

    2. In Figure 2 and 3 where authors used mutagenesis to determine the structural requirements of FL-AR and AR-V7 for nuclear import/retention. These studies used nuclear:cytoplasmic ratios as readouts, not transport kinetics, and thus the observed changes in N/C ratios could be the results of changes in nuclear export and should be discussed appropriately.

    3. The observation that co-expression of AR-V7 increased nulcear FL-AR in the absence of ligand is interesting. The fact that IPZ interferes with nuclear accumulation of FL-AR in the presence of AR-V7 indicated that FL-AR import still requires importin but does not rule out the possibility that FL-AR via its dimerization with AR-V7 within the nucleus could lead to increased retention of FL-AR within the nucleus, a possibility that the authors should consider.

    4. The authors used ChIP assays to confirm the fast chromatin mobility of AR-V7 they have observed using FRAP, however ChIP efficiency could differ significantly using different antibodies and the results should be discussed with caution. Although the authors tried to confirm the same using chromatin bound fraction as another readout for transient chromatin binding of AR-V7, it was unclear why the authors didn't use endogenous AR-V7 in 22RV1 cells to look at chromatin bound fraction, as overexpressed protein may have different behavior compared to endogenous protein.

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