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  • CMS121 br DISCUSSION br The goal of

    2020-08-12


    DISCUSSION
    The goal of this study was to uncover how ARv7 contributes to CRPC development. In this article we show that ARv7 primarily
    Figure 6. ARv7 Represses Genes with a Tumor-Suppressive Function
    (A) GSEA of ARfl- and ARv7-specific gene signatures (Table S5), compared with genes ranked by transcriptome data from CRPC tumors with IHC-defined ARv7 expression. The number of genes in each signature is indicated in parentheses. The normalized enrichment score (NES), the nominal p value (NOM p), and the FDR q value (FDR q) are shown.
    (B) GSEA-determined enrichment profile of the ARv7-repressed gene signature (ARv7 rep) as shown in (A). Genes are ranked by their CMS121 in IHC-defined ARv7 high (red, left, ARv7 pos. corr.) versus low patient tumors (blue, right, ARv7 neg. corr.).
    (C) Heatmap of relative expression of 57 target genes defined by the leading edge in (B), in shGFP and shARv7 LNCaP95 cells. Genes in red were also identified in (D).
    (D) Hockey-stick plot of positively selected genes identified in a genome-wide CRISPR KO screen. Genes were ranked according to their r score and log10 p value. Target genes from the ARv7-repressed gene signature in (A) are indicated, and genes highlighted in red overlap with the leading-edge analysis in (C).
    (E) Hierarchical clustering of the 4 ARv7-target genes from (D) on gene expression data from patient samples (Decipher-GRID). Three clusters, based on the average expression of the four genes, are shown: low (blue), mixed (orange), and high (red).
    (F) Kaplan-Meier graphs of prostate-specific antigen recurrence-free survival for the three patient clusters defined in (E). Log-rank test: n.s, not significant;
    ****p % 0.0001. Box plots show the median, and the first and third quartile. Whiskers extend to 1.5 the interquartile range and data beyond that are shown as individual points.
    acts as a transcriptional repressor, despite colocalization, inter-dependence, and potential heterodimerization of ARv7 with ARfl. This implies that ARv7 and ARfl have divergent transcriptional properties. These differences can be explained, in part, by the structural differences between the two isoforms. Although the N-terminal AF-1 domain harbors substantial transcriptional ac-tivity (Bevan et al., 1999; Ma et al., 1999), the C-terminal LBD, which ARv7 lacks, is necessary for optimal receptor activation. Besides its ability to be bound and activated by hormones, the LBD is also required for the intra- and intermolecular AR N/C-ter-minal interaction, which stabilizes the receptor, regulates core-gulatory interactions (van Royen et al., 2007), and enhances its transactivation function (He et al., 2002). Moreover, the LBD also harbors the capacity to bind LXXLL- and FXXLF-containing coregulators, which in turn modulate the activity of the receptor (Huang et al., 1998; Matias et al., 2000).
    In this work we observed that ARv7 preferentially associates with the NCOR transcriptional corepressors, whereas ARfl asso-ciates with both coactivators and corepressors, in agreement with the idea that ARfl and ARv7 display differential coregulatory binding repertoires. Moreover, increases in ARv7 and NCOR1,2 binding upon ARfl KD suggest that ARfl partially inhibits the ARv7/NCOR interaction, thereby limiting a repressive transcrip-tional response. We find that the difference in ARfl and ARv7 transcriptional activity is correlated with AR isoform-specific dif-ferences in H3K27 acetylation. Therefore, ARv7 likely functions by recruiting corepressors, such as NCOR1 and NCOR2, which in turn control the genomic recruitment of histone deacetylases, such as HDAC3 (Perissi et al., 2010), which negatively regulate H3K27 acetylation. Reprogramming of the FOXA1 cistrome following ARv7 depletion is probably an important consequence of AR inhibition, but may not be directly linked to ARv7-depen-dent repression, since it is also CMS121 observed at ARv7-activated sites. This observation is further supported by the finding that ARv7 and FOXA1 are unable to interact by coIP (He et al., 2018; Wang et al., 2011). Moreover, additional ARv7 cooperating factors may also exist; HOXB13 was recently indicated as an important mediator of ARv7 function (Chen et al., 2018).
    Despite having distinct functions, our findings suggest that ARv7 and ARfl preferentially heterodimerize on chromatin. In contrast to previous studies (Xu et al., 2015), we were unable to verify the presence of ARv7 homodimers by either ChIP-seq or FRET. There are a number of explanations for this discrep-ancy, including the difference between ectopically and endoge-nous expression of ARv7, as found in LNCaP95 cells. We did confirm that ectopically expressed ARv7 alone was able to induce transcription in an AR reporter assay (data not shown). This suggests that the function of ARv7 may differ depending on the abundance of ARfl protein. In general, ARv7 is expressed at much lower levels than ARfl in CRPC (Guo et al., 2009; Robin-son et al., 2015), and no naturally occurring cell lines or tumor models solely expressing ARv7 have been identified. In CRPC cells, ARv7 production is tightly coupled to the transcription rate of the AR gene (Liu et al., 2014), and level of AR gene ampli-fication (Henzler et al., 2016). The presence of large structural rearrangements of the AR locus can uncouple AR-V and ARfl production; it has been previously demonstrated that ARv567es is sufficient to drive CRPC proliferation in the absence of ARfl (Dehm et al., 2008; Nyquist et al., 2013). Therefore, differences