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Cancer Cell. Author manuscript; available in PMC 2017 June 13. Published in final edited form as: Cancer Cell. 2016 June 13; 29(6): 846–858. doi:10.1016/j.ccell.2016.04.012.
TRIM24 is an oncogenic transcriptional activator in prostate cancer
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Anna C. Groner1,2, Laura Cato1,2, Jonas de Tribolet-Hardy1,2, Tiziano Bernasocchi3, Hana Janouskova3, Diana Melchers4, René Houtman4, Andrew C. B. Cato5, Patrick Tschopp6, Lei Gu7, Andrea Corsinotti8,9, Qing Zhong10, Christian Fankhauser10, Christine Fritz10, Cédric Poyet11, Ulrich Wagner10, Tiannan Guo12, Ruedi Aebersold12,13, Levi A. Garraway1,14,15, Peter J. Wild10, Jean-Philippe Theurillat3,14,16,+,*, and Myles Brown1,2,+,* 1Department
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of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA 2Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA 3Institute of Oncology Research, Bellinzona, TI 6500, Switzerland 4PamGene International, Den Bosch, 521HH, The Netherlands 5Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, 76344 EggensteinLeopoldshafen, Germany 6Department of Genetics, Harvard Medical School, Boston, MA 02215, USA 7Division of Newborn Medicine, Children's Hospital Boston and Department of Cell Biology, Harvard Medical School, Boston, MA 02215, USA 8MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh EH16 4UU, Scotland, UK 9Laboratory Animal Resource Center, Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, Japan 10Institute of Surgical Pathology, University Hospital Zurich, Zurich, ZH 8091, Switzerland 11Department of Urology, University Hospital Zurich, Zurich, ZH 8091, Switzerland 12Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, ZH 8093, Switzerland 13Faculty of Science, University of Zurich, Zurich, ZH 8057, Switzerland 14The Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA 15Center for Cancer Genome Discovery, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA 16Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, VD 1011, Switzerland
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*
Correspondence: [email protected]; [email protected]. +These authors contributed equally to this work
Accession Numbers: The GEO accession number for the data series is GSE69332. It includes the microarray data (GSE69330) and the ChIP-seq data (GSE69331). Author Contributions: ACG, JPT, MB conceived and designed the experiments. ACG, LC, TB, HJ, DM performed the experiments. ACG, LC, JDTH, RH, PT, LG, CF, CF, QZ, CP, PJW, JPT analyzed the data. JDTH, LC, RH, ACBC, PT, LG, AC, CF, CF, QZ, CP, UW, TG, RA, PJW, LAG, JPT contributed reagents, materials and analysis tools. ACG, JPT, MB wrote the paper. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Androgen receptor (AR) signaling is a key driver of prostate cancer (PC). While androgendeprivation therapy is transiently effective in advanced disease, tumors often progress to a lethal castration-resistant state (CRPC). We show that recurrent PC-driver mutations in SPOP stabilize the TRIM24 protein, which promotes proliferation under low androgen conditions. TRIM24 augments AR signaling, and AR and TRIM24 co-activated genes are significantly up-regulated in CRPC. Expression of TRIM24 protein increases from primary PC to CRPC, and both TRIM24 protein levels and the AR/TRIM24 gene signature predict disease-recurrence. Analyses in CRPC cells reveal that the TRIM24 bromodomain and the AR-interacting motif are essential to support proliferation. These data provide a rationale for therapeutic TRIM24 targeting in SPOP-mutant and CRPC patients.
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Introduction
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Prostate cancer (PC) remains one of the most common causes of male cancer deaths worldwide (Jemal et al., 2011). Many patients with organ-confined tumors at initial diagnosis relapse following radical prostatectomy or local radiotherapy and develop recurrent disease. On a molecular level, the steroid hormone androgen activates the androgen receptor (AR), which in turn functions as a nuclear receptor transcription factor and executes specific tumorigenic gene expression programs (Matsumoto et al., 2013; Wang et al., 2009). Therefore, advanced cancer therapy includes androgen-deprivation approaches through inhibition of androgen synthesis and the administration of competitive AR antagonists (Niraula et al., 2012). Unfortunately, most patients develop resistance to treatment and subsequently progress to castration-resistant disease (CRPC) that in most cases continues to rely on AR signaling (Heinlein and Chang, 2004; Scher and Sawyers, 2005). As CRPC is often fatal, there is a significant need for improved treatment options. How AR regulates CRPC growth is incompletely characterized, but has been reported to involve mechanisms that enable transactivation of AR under low androgen levels. Proposed processes include intratumoral production of androgens (Montgomery et al., 2008), genetic changes of the AR gene (Taplin et al., 1995; Visakorpi et al., 1995), the emergence of ligand-independent AR splice variants (Guo et al., 2009; Hu et al., 2009; Sun et al., 2010), cross talk between AR and other signaling pathways (Lamont and Tindall, 2011), and the altered action of transcriptional co-regulators (Agoulnik et al., 2006; Gregory et al., 2001; Linja et al., 2004; Taylor et al., 2010; Xu et al., 2012). Genome sequencing studies have revealed recurrent founder mutations in the substratebinding cleft of the cullin-RING ubiquitin ligase adaptor SPOP (speckle-type POZ protein) in approximately 10% of primary PC (Barbieri et al., 2012; Blattner et al., 2014; Kandoth et
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al., 2013). SPOP proteins harboring PC-specific mutations have been reported as being defective in mediating ubiquitylation and proteasomal degradation of AR and its co-activator NCOA3 and thus promote AR signaling (An et al., 2014; Geng et al., 2013; Geng et al., 2014). In agreement with this, enhanced AR signaling has been identified as a cardinal feature of SPOP mutant tumors (TCGA, 2015). Using an unbiased proteomic approach in prostate epithelial cells, we identified TRIM24 (tripartite motif-containing protein 24, also known as TIF1α) as another potential effector protein downstream of SPOP mutations (Theurillat et al., 2014). Moreover, TRIM24 showed reduced ubiquitylation that was accompanied by increased protein levels in the presence of SPOP mutations (Theurillat et al., 2014).
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TRIM24 has been implicated in driving different tumor types through its ability to interfere with tumor suppressive and oncogenic pathways (Hatakeyama, 2011; Herquel et al., 2011). Its N-terminal tripartite motifs include a RING domain, which is involved in ubiquitylation and degradation of p53 (Allton et al., 2009). Moreover, a C-terminal tandem PHD fingerbromodomain confers TRIM24 with the ability to recognize unmodified histone H3K4 through the former domain, as well as H3K23-acetyl through the latter domain (Tsai et al., 2010). This chromatin interacting module has been implicated in general transcriptional coregulation, as well as the activation of estrogen-responsive genes in breast cancer and the PIK3CA gene in glioma (Herquel et al., 2011; Tsai et al., 2010; Zhang et al., 2015). Through its conserved single LxxLL motif TRIM24 interacts with the AF2 domain of several nuclear receptors, including AR (Le Douarin et al., 1996; Thenot et al., 1997; vom Baur et al., 1996). In line with these observations, TRIM24 was shown to enhance AR-mediated gene activation in reporter assays (Kikuchi et al., 2009). Whether these different functions of TRIM24 influence PC progression is not known.
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In this study we sought to investigate the role of TRIM24 in PC and to elucidate its suitability as a therapeutic target by combining a molecular characterization in PC cell lines with analyses in PC patients. We hypothesized that TRIM24 may be important for PC progression by functioning as an oncogenic transcriptional activator that cooperates with AR-dependent gene expression in CRPC settings, potentially opening new therapeutic avenues for treatment.
Results TRIM24 mediates SPOP-mutant PC cell proliferation in low androgen
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To investigate the impact of PC-associated SPOP mutants on TRIM24 deregulation and on androgen-mediated cell proliferation, we tested the effect of different SPOP mutations in the androgen-dependent LNCaP cell line. We observed that levels of TRIM24 protein were augmented upon expression of a number of different PC-associated SPOP mutations (Figure 1A), which is in agreement with our earlier results in prostate epithelial cells (Theurillat et al., 2014). Importantly, TRIM24 mRNA levels that were assessed in a parallel experiment did not follow the protein expression changes (Figure 1B), suggesting that these SPOP mutations regulate TRIM24 at the protein level in LNCaP cells. To examine how SPOP affects TRIM24 protein stability in LNCaP cells, we made use of a HA-tagged form of TRIM24 and found that this protein decayed more rapidly in the presence of wild-type (WT) Cancer Cell. Author manuscript; available in PMC 2017 June 13.
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SPOP than with the SPOP-F133L mutant (Figure S1A). In agreement with TRIM24 being an SPOP target, we found that SPOP-WT expression and treatment with the proteasome inhibitor MG132 increased TRIM24 ubiquitylation in 293T cells (Figure S1B), and SPOP knockdown augmented TRIM24 protein levels in LNCaP cells (Figure S1C). These results are consistent with our earlier findings in prostate epithelial cells (Theurillat et al., 2014) and support a model in which SPOP mutants impair ubiquitylation and degradation of TRIM24 through the proteasome.
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Next we measured androgen-dependent LNCaP growth, in the presence of SPOP mutants, across a range of androgen concentrations. We found that when only limiting amounts of 5α-dihydrotestosterone (DHT) were available, SPOP mutant-expressing LNCaP cells all displayed a significant growth advantage compared to cells expressing SPOP-WT (Figures 1Ca and S1D). This growth advantage seen in SPOP-Y87C expressing cells cultured under low DHT concentrations was abrogated when TRIM24 expression was silenced by a specific shRNA (Figures 1Cb and S1E). The SPOP-W131G mutant showed comparable results (Figures S1F and S1G). At a higher DHT concentration, silencing of TRIM24 partially decreased the SPOP-Y87C mediated growth advantage (Figure 1Cb), suggesting that additional proteins may contribute to the SPOP mutant phenotype. This is also reflected in the DHT-induced values of proliferation, expressed as the half maximal effective concentration (EC50). The EC50 for the SPOP-Y87C mutant cells is 3-fold lower than the EC50 of SPOP-WT cells, while knock-down of TRIM24 in the mutant cells does not fully revert back to the WT EC50 level (Figure S1H). In summary, these findings reveal that the stabilization of TRIM24 protein by SPOP mutations is necessary to promote optimal PC proliferation under low androgen conditions.
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TRIM24 promotes PC growth and sensitizes cells to low androgen availability
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We then asked whether TRIM24 is sufficient to promote PC growth under low androgen availability. Indeed, over-expression of doxycycline (dox)-inducible TRIM24 (Figure S1I) was sufficient to mediate increased proliferation of LNCaP cells at 0.1nM DHT, when compared to GFP expressing control cells (Figure 1D). Conversely, when we decreased endogenous TRIM24 levels with shRNA (Figure S1J), we saw a reduction in androgendependent LNCaP proliferation (Figure 1Ea). Again, this phenotype was most prominent under low DHT concentrations (Figure 1Eb). Therefore, we tested the effect of TRIM24 depletion on the growth of the LNCaP-derived CRPC line LNCaP-abl (abl), which is continuously cultured in the absence of androgens. We found that reduction of TRIM24 with 4 different shRNA-based hairpins (Figure S1K) resulted in impaired proliferation rates for each of them (Figure 1F). More specifically, TRIM24-depleted abl cells exhibited a decreased capacity of G1/S cell cycle transition, without an increase in apoptosis (Figures S1L-S1O). In addition, TRIM24 knock-down triggered similar phenotypes in the androgenindependent lines CWR-22Rv1 and LNCaP95 (Figures S1P and S1Q), further supporting the conclusion that TRIM24 is essential for PC proliferation in castration-resistant settings.
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TRIM24 binds to promoters and activates genes involved in cell proliferation and AR signaling in PC cells
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To understand why hormone-starved PC cells were more dependent on TRIM24 than hormone-stimulated cells, we set out to identify TRIM24-dependent gene expression programs by overlapping TRIM24 cistromes with TRIM24-regulated genes. Direct genomic binding of TRIM24 was determined by performing ChIP-seq in abl as well as in LNCaP cells cultured in the absence or presence of hormone stimulation (Figure 2A). TRIM24 was found to be most abundant near promoters in all cistromes (Figures 2B and 2C). The number of TRIM24 sites went from 9744 and 7105 in hormone-starved and hormone-stimulated LNCaP cells, respectively, to 20621 sites in abl cells, with most LNCaP-specific TRIM24 sites also present in abl cells (Figure 2A). In addition, most of the over-represented transcription factor binding motifs associated with the TRIM24 cistromes are shared in both LNCaP and abl cells (Table S1). We also assessed the overlap of the LNCaP-specific TRIM24 ChIP-seq with published H3K27-acetyl data sets (Hazelett et al., 2014), which is a histone mark to which the TRIM24 bromodomain binds (Tsai et al., 2010). We identified a significant overlap between the respective cistromes both in the absence or presence of hormone-stimulation (Figures 2D and 2E, p2), consistent with TRIM24 acting as a transcriptional activator in this setting (Figure S2A). Moreover, 107 and 117 genes showed decreased levels after TRIM24 knockdown in LNCaP cells cultured without or with DHT stimulation, respectively (Figure S2A). In abl cells 278 genes were down-regulated upon TRIM24 depletion (Figure S2A). Many TRIM24-activated genes were involved in cell cycle processes (Figure S2B) and were up-regulated in abl cells compared to LNCaP cells (Figures S2C and S2D). Therefore, TRIM24 more prominently activates cell cycle genes in abl cells, thus providing an explanation for the increased dependency of these CRPC cells on intact TRIM24 levels for proliferation.
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The correlation of the TRIM24 cistromes with TRIM24-regulated genes revealed a significant association between TRIM24 binding and transcriptional activation in both LNCaP and abl cells (Figure S2E). We further validated a set of TRIM24-activated gene targets by ChIP-quantitative PCR (qPCR) (Figures S2F and S2G) and Western blotting (Figure S2H). We then determined direct TRIM24-activated targets by identifying genes with promoter-bound TRIM24 (3kb around TSS) that were also significantly down-regulated by TRIM24 depletion (LIMMA, p1.3). To enable pathway analyses on the direct TRIM24-activated gene targets, we enlarged the target gene lists using a less stringent fold-change cutoff. In this analysis, 438 and 409 TRIM24 target genes were identified in LNCaP cells cultured under hormone-starvation (-DHT) and -stimulation (+DHT), respectively (Figure 2F and Table S2). In addition, we also found 1218 genes that were positively regulated by TRIM24 binding by applying the same criteria to the abl cell data sets (Figure 2F and Table S2).
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We then used gene set enrichment analyses to compare the identified TRIM24-activated gene targets with the curated HALLMARK gene sets present in the Molecular Signatures Database (MSigDB) (Subramanian et al., 2005). When we determined the top 10 most enriched biological processes, 5 of them were common among all the different TRIM24activated target sets (Figure 2F and Table S3M). We then focused on identifying differences in the prevalence of genes present in the common pathways that would help explain our phenotypic findings in the different PC cell lines. In line with TRIM24 driving abl cell proliferation (Figure 1F), we found that cell cycle-related pathways, such as the G2M checkpoint and E2F targets were more prominently regulated in these CRPC cells compared to LNCaP cells (Figure 2F). Androgen responsive genes were more frequently activated by TRIM24 in LNCaP and abl cells under hormone-starved conditions, when compared to hormone-stimulated LNCaP cells (Figure 2F). This result is consistent with TRIM24 mediating PC cell growth under low DHT levels (Figures 1D and 1E) and suggests that TRIM24 co-activates AR-regulated genes more prominently under limiting hormone conditions and is thus more essential for PC proliferation in the castration-resistant setting.
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To assess if TRIM24 and AR work together to co-activate target genes, we explored the interaction between AR and TRIM24. First, we found a significant overlap between the AR and the TRIM24 cistromes (Figures S3A and S3B) with a DHT-dependent increase of TRIM24 binding at the shared sites in LNCaP cells (Figure S3C). Second, we could confirm a weak interaction between the endogenous proteins when performing AR-specific immunoprecipitation on nuclear extracts (Figure S3D). Finally, by using peptide arrays (Figure S3E), we also validated the reported interaction between the TRIM24 LxxLL motif and the AR ligand binding domain (Cavailles et al., 1995; Kikuchi et al., 2009; Le Douarin et al., 1996; Thenot et al., 1997). Taken together, these results imply that AR and TRIM24 directly cooperate to activate genes in PC though we cannot exclude participation by another LxxLL-binding factor in the complex. Moreover, the overlap between AR and TRIM24 genomic binding is more pronounced in abl cells (2419 sites) when compared to LNCaP cells (526 sites) (Figures S3A and S3B), consistent with an increased interaction between the two factors in CRPC. AR and TRIM24 directly activate genes that are up-regulated in CRPC and predict recurrence in primary tumors
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To assess which genes are co-activated by AR and TRIM24 in CRPC cells, we derived an AR/TRIM24-regulated signature consisting of 21 genes using cistrome and gene expression data from abl cells. More specifically, genes within the signature were defined as those in a 100kb interval around overlapping AR and TRIM24 sites, which were also down-regulated by siRNA targeting AR and TRIM24 (LIMMA, p1.5). We could detect concomitant recruitment of AR and TRIM24 on the same allele by ChIP-reChIP for the AURKB enhancer and the PBK promoter (Figures S3F and S3G). We then used this signature to stratify publicly available gene expression data from PC patients (Taylor et al., 2010) and identified two prominent clusters (Figure 3A). When we calculated the average expression of all AR/TRIM24 targets for the two clusters, we found that one cluster showed a significantly higher average gene expression when compared to the other cluster (Figure 3B). Thus, we named the clusters “low” (grey) and “high” (yellow). The “high” cluster was
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significantly enriched in metastatic samples compared to the “low” cluster (Figure 3A, fisher exact test, p
Cancer Cell. Author manuscript; available in PMC 2017 June 13. Published in final edited form as: Cancer Cell. 2016 June 13; 29(6): 846–858. doi:10.1016/j.ccell.2016.04.012.
TRIM24 is an oncogenic transcriptional activator in prostate cancer
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Anna C. Groner1,2, Laura Cato1,2, Jonas de Tribolet-Hardy1,2, Tiziano Bernasocchi3, Hana Janouskova3, Diana Melchers4, René Houtman4, Andrew C. B. Cato5, Patrick Tschopp6, Lei Gu7, Andrea Corsinotti8,9, Qing Zhong10, Christian Fankhauser10, Christine Fritz10, Cédric Poyet11, Ulrich Wagner10, Tiannan Guo12, Ruedi Aebersold12,13, Levi A. Garraway1,14,15, Peter J. Wild10, Jean-Philippe Theurillat3,14,16,+,*, and Myles Brown1,2,+,* 1Department
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of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA 2Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA 3Institute of Oncology Research, Bellinzona, TI 6500, Switzerland 4PamGene International, Den Bosch, 521HH, The Netherlands 5Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, 76344 EggensteinLeopoldshafen, Germany 6Department of Genetics, Harvard Medical School, Boston, MA 02215, USA 7Division of Newborn Medicine, Children's Hospital Boston and Department of Cell Biology, Harvard Medical School, Boston, MA 02215, USA 8MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh EH16 4UU, Scotland, UK 9Laboratory Animal Resource Center, Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, Japan 10Institute of Surgical Pathology, University Hospital Zurich, Zurich, ZH 8091, Switzerland 11Department of Urology, University Hospital Zurich, Zurich, ZH 8091, Switzerland 12Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, ZH 8093, Switzerland 13Faculty of Science, University of Zurich, Zurich, ZH 8057, Switzerland 14The Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA 15Center for Cancer Genome Discovery, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA 16Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, VD 1011, Switzerland
Summary Author Manuscript
*
Correspondence: [email protected]; [email protected]. +These authors contributed equally to this work
Accession Numbers: The GEO accession number for the data series is GSE69332. It includes the microarray data (GSE69330) and the ChIP-seq data (GSE69331). Author Contributions: ACG, JPT, MB conceived and designed the experiments. ACG, LC, TB, HJ, DM performed the experiments. ACG, LC, JDTH, RH, PT, LG, CF, CF, QZ, CP, PJW, JPT analyzed the data. JDTH, LC, RH, ACBC, PT, LG, AC, CF, CF, QZ, CP, UW, TG, RA, PJW, LAG, JPT contributed reagents, materials and analysis tools. ACG, JPT, MB wrote the paper. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Groner et al.
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Androgen receptor (AR) signaling is a key driver of prostate cancer (PC). While androgendeprivation therapy is transiently effective in advanced disease, tumors often progress to a lethal castration-resistant state (CRPC). We show that recurrent PC-driver mutations in SPOP stabilize the TRIM24 protein, which promotes proliferation under low androgen conditions. TRIM24 augments AR signaling, and AR and TRIM24 co-activated genes are significantly up-regulated in CRPC. Expression of TRIM24 protein increases from primary PC to CRPC, and both TRIM24 protein levels and the AR/TRIM24 gene signature predict disease-recurrence. Analyses in CRPC cells reveal that the TRIM24 bromodomain and the AR-interacting motif are essential to support proliferation. These data provide a rationale for therapeutic TRIM24 targeting in SPOP-mutant and CRPC patients.
Graphical abstract Author Manuscript
Introduction
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Prostate cancer (PC) remains one of the most common causes of male cancer deaths worldwide (Jemal et al., 2011). Many patients with organ-confined tumors at initial diagnosis relapse following radical prostatectomy or local radiotherapy and develop recurrent disease. On a molecular level, the steroid hormone androgen activates the androgen receptor (AR), which in turn functions as a nuclear receptor transcription factor and executes specific tumorigenic gene expression programs (Matsumoto et al., 2013; Wang et al., 2009). Therefore, advanced cancer therapy includes androgen-deprivation approaches through inhibition of androgen synthesis and the administration of competitive AR antagonists (Niraula et al., 2012). Unfortunately, most patients develop resistance to treatment and subsequently progress to castration-resistant disease (CRPC) that in most cases continues to rely on AR signaling (Heinlein and Chang, 2004; Scher and Sawyers, 2005). As CRPC is often fatal, there is a significant need for improved treatment options. How AR regulates CRPC growth is incompletely characterized, but has been reported to involve mechanisms that enable transactivation of AR under low androgen levels. Proposed processes include intratumoral production of androgens (Montgomery et al., 2008), genetic changes of the AR gene (Taplin et al., 1995; Visakorpi et al., 1995), the emergence of ligand-independent AR splice variants (Guo et al., 2009; Hu et al., 2009; Sun et al., 2010), cross talk between AR and other signaling pathways (Lamont and Tindall, 2011), and the altered action of transcriptional co-regulators (Agoulnik et al., 2006; Gregory et al., 2001; Linja et al., 2004; Taylor et al., 2010; Xu et al., 2012). Genome sequencing studies have revealed recurrent founder mutations in the substratebinding cleft of the cullin-RING ubiquitin ligase adaptor SPOP (speckle-type POZ protein) in approximately 10% of primary PC (Barbieri et al., 2012; Blattner et al., 2014; Kandoth et
Cancer Cell. Author manuscript; available in PMC 2017 June 13.
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al., 2013). SPOP proteins harboring PC-specific mutations have been reported as being defective in mediating ubiquitylation and proteasomal degradation of AR and its co-activator NCOA3 and thus promote AR signaling (An et al., 2014; Geng et al., 2013; Geng et al., 2014). In agreement with this, enhanced AR signaling has been identified as a cardinal feature of SPOP mutant tumors (TCGA, 2015). Using an unbiased proteomic approach in prostate epithelial cells, we identified TRIM24 (tripartite motif-containing protein 24, also known as TIF1α) as another potential effector protein downstream of SPOP mutations (Theurillat et al., 2014). Moreover, TRIM24 showed reduced ubiquitylation that was accompanied by increased protein levels in the presence of SPOP mutations (Theurillat et al., 2014).
Author Manuscript
TRIM24 has been implicated in driving different tumor types through its ability to interfere with tumor suppressive and oncogenic pathways (Hatakeyama, 2011; Herquel et al., 2011). Its N-terminal tripartite motifs include a RING domain, which is involved in ubiquitylation and degradation of p53 (Allton et al., 2009). Moreover, a C-terminal tandem PHD fingerbromodomain confers TRIM24 with the ability to recognize unmodified histone H3K4 through the former domain, as well as H3K23-acetyl through the latter domain (Tsai et al., 2010). This chromatin interacting module has been implicated in general transcriptional coregulation, as well as the activation of estrogen-responsive genes in breast cancer and the PIK3CA gene in glioma (Herquel et al., 2011; Tsai et al., 2010; Zhang et al., 2015). Through its conserved single LxxLL motif TRIM24 interacts with the AF2 domain of several nuclear receptors, including AR (Le Douarin et al., 1996; Thenot et al., 1997; vom Baur et al., 1996). In line with these observations, TRIM24 was shown to enhance AR-mediated gene activation in reporter assays (Kikuchi et al., 2009). Whether these different functions of TRIM24 influence PC progression is not known.
Author Manuscript
In this study we sought to investigate the role of TRIM24 in PC and to elucidate its suitability as a therapeutic target by combining a molecular characterization in PC cell lines with analyses in PC patients. We hypothesized that TRIM24 may be important for PC progression by functioning as an oncogenic transcriptional activator that cooperates with AR-dependent gene expression in CRPC settings, potentially opening new therapeutic avenues for treatment.
Results TRIM24 mediates SPOP-mutant PC cell proliferation in low androgen
Author Manuscript
To investigate the impact of PC-associated SPOP mutants on TRIM24 deregulation and on androgen-mediated cell proliferation, we tested the effect of different SPOP mutations in the androgen-dependent LNCaP cell line. We observed that levels of TRIM24 protein were augmented upon expression of a number of different PC-associated SPOP mutations (Figure 1A), which is in agreement with our earlier results in prostate epithelial cells (Theurillat et al., 2014). Importantly, TRIM24 mRNA levels that were assessed in a parallel experiment did not follow the protein expression changes (Figure 1B), suggesting that these SPOP mutations regulate TRIM24 at the protein level in LNCaP cells. To examine how SPOP affects TRIM24 protein stability in LNCaP cells, we made use of a HA-tagged form of TRIM24 and found that this protein decayed more rapidly in the presence of wild-type (WT) Cancer Cell. Author manuscript; available in PMC 2017 June 13.
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SPOP than with the SPOP-F133L mutant (Figure S1A). In agreement with TRIM24 being an SPOP target, we found that SPOP-WT expression and treatment with the proteasome inhibitor MG132 increased TRIM24 ubiquitylation in 293T cells (Figure S1B), and SPOP knockdown augmented TRIM24 protein levels in LNCaP cells (Figure S1C). These results are consistent with our earlier findings in prostate epithelial cells (Theurillat et al., 2014) and support a model in which SPOP mutants impair ubiquitylation and degradation of TRIM24 through the proteasome.
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Next we measured androgen-dependent LNCaP growth, in the presence of SPOP mutants, across a range of androgen concentrations. We found that when only limiting amounts of 5α-dihydrotestosterone (DHT) were available, SPOP mutant-expressing LNCaP cells all displayed a significant growth advantage compared to cells expressing SPOP-WT (Figures 1Ca and S1D). This growth advantage seen in SPOP-Y87C expressing cells cultured under low DHT concentrations was abrogated when TRIM24 expression was silenced by a specific shRNA (Figures 1Cb and S1E). The SPOP-W131G mutant showed comparable results (Figures S1F and S1G). At a higher DHT concentration, silencing of TRIM24 partially decreased the SPOP-Y87C mediated growth advantage (Figure 1Cb), suggesting that additional proteins may contribute to the SPOP mutant phenotype. This is also reflected in the DHT-induced values of proliferation, expressed as the half maximal effective concentration (EC50). The EC50 for the SPOP-Y87C mutant cells is 3-fold lower than the EC50 of SPOP-WT cells, while knock-down of TRIM24 in the mutant cells does not fully revert back to the WT EC50 level (Figure S1H). In summary, these findings reveal that the stabilization of TRIM24 protein by SPOP mutations is necessary to promote optimal PC proliferation under low androgen conditions.
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TRIM24 promotes PC growth and sensitizes cells to low androgen availability
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We then asked whether TRIM24 is sufficient to promote PC growth under low androgen availability. Indeed, over-expression of doxycycline (dox)-inducible TRIM24 (Figure S1I) was sufficient to mediate increased proliferation of LNCaP cells at 0.1nM DHT, when compared to GFP expressing control cells (Figure 1D). Conversely, when we decreased endogenous TRIM24 levels with shRNA (Figure S1J), we saw a reduction in androgendependent LNCaP proliferation (Figure 1Ea). Again, this phenotype was most prominent under low DHT concentrations (Figure 1Eb). Therefore, we tested the effect of TRIM24 depletion on the growth of the LNCaP-derived CRPC line LNCaP-abl (abl), which is continuously cultured in the absence of androgens. We found that reduction of TRIM24 with 4 different shRNA-based hairpins (Figure S1K) resulted in impaired proliferation rates for each of them (Figure 1F). More specifically, TRIM24-depleted abl cells exhibited a decreased capacity of G1/S cell cycle transition, without an increase in apoptosis (Figures S1L-S1O). In addition, TRIM24 knock-down triggered similar phenotypes in the androgenindependent lines CWR-22Rv1 and LNCaP95 (Figures S1P and S1Q), further supporting the conclusion that TRIM24 is essential for PC proliferation in castration-resistant settings.
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TRIM24 binds to promoters and activates genes involved in cell proliferation and AR signaling in PC cells
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To understand why hormone-starved PC cells were more dependent on TRIM24 than hormone-stimulated cells, we set out to identify TRIM24-dependent gene expression programs by overlapping TRIM24 cistromes with TRIM24-regulated genes. Direct genomic binding of TRIM24 was determined by performing ChIP-seq in abl as well as in LNCaP cells cultured in the absence or presence of hormone stimulation (Figure 2A). TRIM24 was found to be most abundant near promoters in all cistromes (Figures 2B and 2C). The number of TRIM24 sites went from 9744 and 7105 in hormone-starved and hormone-stimulated LNCaP cells, respectively, to 20621 sites in abl cells, with most LNCaP-specific TRIM24 sites also present in abl cells (Figure 2A). In addition, most of the over-represented transcription factor binding motifs associated with the TRIM24 cistromes are shared in both LNCaP and abl cells (Table S1). We also assessed the overlap of the LNCaP-specific TRIM24 ChIP-seq with published H3K27-acetyl data sets (Hazelett et al., 2014), which is a histone mark to which the TRIM24 bromodomain binds (Tsai et al., 2010). We identified a significant overlap between the respective cistromes both in the absence or presence of hormone-stimulation (Figures 2D and 2E, p2), consistent with TRIM24 acting as a transcriptional activator in this setting (Figure S2A). Moreover, 107 and 117 genes showed decreased levels after TRIM24 knockdown in LNCaP cells cultured without or with DHT stimulation, respectively (Figure S2A). In abl cells 278 genes were down-regulated upon TRIM24 depletion (Figure S2A). Many TRIM24-activated genes were involved in cell cycle processes (Figure S2B) and were up-regulated in abl cells compared to LNCaP cells (Figures S2C and S2D). Therefore, TRIM24 more prominently activates cell cycle genes in abl cells, thus providing an explanation for the increased dependency of these CRPC cells on intact TRIM24 levels for proliferation.
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The correlation of the TRIM24 cistromes with TRIM24-regulated genes revealed a significant association between TRIM24 binding and transcriptional activation in both LNCaP and abl cells (Figure S2E). We further validated a set of TRIM24-activated gene targets by ChIP-quantitative PCR (qPCR) (Figures S2F and S2G) and Western blotting (Figure S2H). We then determined direct TRIM24-activated targets by identifying genes with promoter-bound TRIM24 (3kb around TSS) that were also significantly down-regulated by TRIM24 depletion (LIMMA, p1.3). To enable pathway analyses on the direct TRIM24-activated gene targets, we enlarged the target gene lists using a less stringent fold-change cutoff. In this analysis, 438 and 409 TRIM24 target genes were identified in LNCaP cells cultured under hormone-starvation (-DHT) and -stimulation (+DHT), respectively (Figure 2F and Table S2). In addition, we also found 1218 genes that were positively regulated by TRIM24 binding by applying the same criteria to the abl cell data sets (Figure 2F and Table S2).
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We then used gene set enrichment analyses to compare the identified TRIM24-activated gene targets with the curated HALLMARK gene sets present in the Molecular Signatures Database (MSigDB) (Subramanian et al., 2005). When we determined the top 10 most enriched biological processes, 5 of them were common among all the different TRIM24activated target sets (Figure 2F and Table S3M). We then focused on identifying differences in the prevalence of genes present in the common pathways that would help explain our phenotypic findings in the different PC cell lines. In line with TRIM24 driving abl cell proliferation (Figure 1F), we found that cell cycle-related pathways, such as the G2M checkpoint and E2F targets were more prominently regulated in these CRPC cells compared to LNCaP cells (Figure 2F). Androgen responsive genes were more frequently activated by TRIM24 in LNCaP and abl cells under hormone-starved conditions, when compared to hormone-stimulated LNCaP cells (Figure 2F). This result is consistent with TRIM24 mediating PC cell growth under low DHT levels (Figures 1D and 1E) and suggests that TRIM24 co-activates AR-regulated genes more prominently under limiting hormone conditions and is thus more essential for PC proliferation in the castration-resistant setting.
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To assess if TRIM24 and AR work together to co-activate target genes, we explored the interaction between AR and TRIM24. First, we found a significant overlap between the AR and the TRIM24 cistromes (Figures S3A and S3B) with a DHT-dependent increase of TRIM24 binding at the shared sites in LNCaP cells (Figure S3C). Second, we could confirm a weak interaction between the endogenous proteins when performing AR-specific immunoprecipitation on nuclear extracts (Figure S3D). Finally, by using peptide arrays (Figure S3E), we also validated the reported interaction between the TRIM24 LxxLL motif and the AR ligand binding domain (Cavailles et al., 1995; Kikuchi et al., 2009; Le Douarin et al., 1996; Thenot et al., 1997). Taken together, these results imply that AR and TRIM24 directly cooperate to activate genes in PC though we cannot exclude participation by another LxxLL-binding factor in the complex. Moreover, the overlap between AR and TRIM24 genomic binding is more pronounced in abl cells (2419 sites) when compared to LNCaP cells (526 sites) (Figures S3A and S3B), consistent with an increased interaction between the two factors in CRPC. AR and TRIM24 directly activate genes that are up-regulated in CRPC and predict recurrence in primary tumors
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To assess which genes are co-activated by AR and TRIM24 in CRPC cells, we derived an AR/TRIM24-regulated signature consisting of 21 genes using cistrome and gene expression data from abl cells. More specifically, genes within the signature were defined as those in a 100kb interval around overlapping AR and TRIM24 sites, which were also down-regulated by siRNA targeting AR and TRIM24 (LIMMA, p1.5). We could detect concomitant recruitment of AR and TRIM24 on the same allele by ChIP-reChIP for the AURKB enhancer and the PBK promoter (Figures S3F and S3G). We then used this signature to stratify publicly available gene expression data from PC patients (Taylor et al., 2010) and identified two prominent clusters (Figure 3A). When we calculated the average expression of all AR/TRIM24 targets for the two clusters, we found that one cluster showed a significantly higher average gene expression when compared to the other cluster (Figure 3B). Thus, we named the clusters “low” (grey) and “high” (yellow). The “high” cluster was
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significantly enriched in metastatic samples compared to the “low” cluster (Figure 3A, fisher exact test, p
