TRIM24 Is an Oncogenic Transcriptional Activator in Prostate Cancer

Abstract

Androgen receptor (AR) signaling is a key driver of prostate cancer (PC). While androgen-deprivation 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 speckle-type POZ protein (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 upregulated 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.

Figure 1. Mutant SPOP stabilizes TRIM24 promoting PC cell growth under low androgen

(A) TRIM24 and SPOP protein levels were detected by Western blot in LNCaP cells stably expressing wild-type (WT) or the indicated mutant SPOP constructs (in grey). Calnexin was used as a loading control. (B) TRIM24 protein levels from the indicated SPOP lines were compared to TRIM24 mRNA as assessed by quantitative PCR. (C) LNCaP cells stably expressing SPOP-WT or SPOP-Y87C were infected with lentiviral doxycycline (dox)-inducible shRNA targeting TRIM24 or a non-targeting (NT) control. Cells were treated with dox while being grown in indicated DHT concentrations and counted by hemacytometer (n=3) after 6 days. (a) Cell numbers were plotted against a log₁₀ scale of the DHT concentration. (b) Cell numbers under low DHT levels are additionally highlighted in a bar plot. (D) LNCaP cells harboring dox-inducible TRIM24 or GFP cDNA were treated with dox for 6 days, while being grown in the presence of the indicated DHT concentration. Cell numbers were assessed as in (C). (E) LNCaP cells stably expressing dox-inducible shRNA targeting TRIM24 or a NT control were treated with dox for 6 days while being grown in the presence of the indicated concentration of DHT. Cell numbers were counted as in (C). (a) Cell numbers were plotted against a log₁₀ scale of the DHT concentration. (b) Cell numbers under low DHT levels are additionally highlighted in a bar plot. (F) LNCaP-abl cells stably expressing 4 different dox-inducible shRNAs targeting TRIM24 or a NT control were treated with dox at day 0 and grown for 9 days. Cell numbers were counted after the indicated days and plotted. TRIM24 protein levels were assessed at the end of each experiment and are shown in Figures S1E, S1I, S1J and S1K. Data are represented as mean +/- SEM. Statistical analysis was performed using a two-tailed student's t-test assuming unequal variance *p<0.05, ** p<0.01, *** p<0.005. (See also Figure S1).

Figure 2. TRIM24 binds to promoters and activates genes involved in cell proliferation and AR signaling in PC cells

(A) Venn diagram overlapping the TRIM24 cistromes determined in hormone-starved (-DHT) and hormone-stimulated (+DHT) LNCaP (LN) cells, in addition to the hormone-refractory line LNCaP-abl (abl). The total number of genomic binding sites for each cistrome is indicated in parenthesis. (B) TRIM24 ChIP-seq signals from (A) were expressed relative to the location of TSS in the genome. (C) Cis-regulatory Element Annotation System (CEAS) (Ji et al., 2006) was used to characterize the genome-wide binding of the TRIM24-specific cistromes. Assessed binding distribution included regions up to 3kb upstream of TSS (promoter), up to 3kb downstream of TSS (downstream), 5′ and 3′ UTRs, coding exons, introns and distal intergenic regions. (D, E) Venn diagrams overlapping TRIM24 cistromes with published Histone H3 K27-acetylation (H3K27ac) cistromes (Hazelett et al., 2014) reveal (D) a 5266 site overlap in LN -DHT (p<0.001, random permutation), and (E) a 4307 site overlap in LN +DHT (p<0.001, random permutation). The total number of genomic binding sites for each cistrome is indicated in parenthesis. (F) Direct TRIM24-activated gene targets were determined by overlapping TRIM24 cistromes with TRIM24-dependent gene expression profiles in LN cells with our without DHT-stimulation and in abl cells. Direct targets were defined by TRIM24 promoter binding (3kb around TSS) and significant siRNA-mediated down-regulation (LIMMA, p<0.05, fold-change>1.3). Gene set enrichment analysis was used to compare TRIM24 targets with curated HALLMARK gene sets (Molecular Signatures Database). The top 5 common pathways together with their significance values (-log p-value) are depicted. (See also Figure S2 and Tables S1-S3).

Figure 3. AR and TRIM24 co-activated genes are up-regulated in CRPC and predict disease recurrence in primary tumors

(A) AR/TRIM24 co-activated targets in CRPC were defined as the 21 genes bound by overlapping AR and TRIM24 sites, within 100kb of their TSS and down-regulated by siRNA-mediated knock-down of both factors in abl cells (LIMMA, p<0.05, fold-change>1.5). Hierarchical clustering of patient gene expression data (MSKCC/Taylor set) (Taylor et al., 2010) with the 21-gene signature resulted in two main clusters indicated in grey and yellow, respectively. The clusters were named based on the average expression of the 21-gene signature, which was lower in the grey cluster (low) compared to the yellow cluster (high). The metastatic samples in the data set are marked in light blue. (B) Average expression levels of the 21 AR/TRIM24 gene targets are depicted for both clusters. Data are represented as mean +/- SEM. Statistical analysis was performed using a two-tailed student's t-test assuming unequal variance p<0.001. (C) Kaplan-Meier curve for PSA-recurrence-free survival. Patients were stratified as high and low according to the 21-gene expression signature in primary tumors (p-value, log rank test). p<0.001. (See also Figure S3).

Figure 4. TRIM24 protein levels increase during PC progression and predict disease recurrence in primary tumors

(A) TRIM24 immunohistochemistry (IHC) analysis on tumor tissue microarrays (TMA) of benign prostate hyperplasia (BPH), primary PC tumors (PPC), and advanced castration-resistant disease (CRPC) using a three-tiered scoring system as previously described (Theurillat et al., 2014). Bar represents 50 mm. (B) Quantification and percentage of negative, weak and strong TRIM24 nuclear staining of patients with BPH, PPC, and CRPC. (C) Kaplan-Meier curves with pointwise 95% confidence bands of PSA-based recurrence-free survival. Patients were stratified as negative, weak or strong TRIM24 nuclear staining (p-value, log rank test, p<0.001). Patients with primary PC undergoing radical prostatectomy and reaching the PSA nadir (<0.1ng/mL) postoperatively were used for analysis. (See also Figure S4 and Table S4)

Figure 5. Univariate and multivariate Cox regression analysis on a primary PC cohort

(A, B) Univariate (A) and multivariate (B) Cox regression analyses of the association between patients' characteristics at surgery and the probability of recurrence-free survival based on PSA levels. Patients' characteristics included age, BMI, Gleason score, tumor size (pT), metastatic spread to local lymph nodes (pN), margin status at resection, PSA at diagnosis, SPOP mutation status, NCOA3, AR and TRIM24 staining. TRIM24 trichotomy and dichotomy are defined as follows: TRIM24 trichotomy includes three-tiers corresponding to negative, weak or strong TRIM24 expression. TRIM24 dichotomy includes two-tiers where negative includes negative or weak TRIM24 expression and positive includes strong expression only. Time to PSA recurrence (cut-off 0.1ng/mL) was selected as clinical end point. The hazard ratios were estimated with a univariate (A) or multivariate (B) Cox proportional hazards model. The dashed vertical line was drawn at the no effect point (hazard ratio of 1.0). Horizontal lines represent a 95% confidence interval. The mid-point of the box represents the mean effect estimate and the area of the box represents the weight for each subgroup. Significant p-values (p<0.05) are marked in bold. Limit for reverse selection procedure was p=0.1.

Figure 6. TRIM24 over-expression accelerates intraprostatic tumor cell growth in mice

(A-C) LNCaP (LN) cells harboring constitutive expression of GFP or TRIM24 were grown (A) in the presence of the indicated concentration of DHT for 6 days or (B) in full media for 5 days and counted by hemacytometer (n=3). (C) TRIM24 protein levels were measured by Western blotting. Modified cells were either cultured in the presence of the indicated concentration of DHT or in full media (full). GAPDH served as a loading control. (D-G) Intraprostatic injection of modified LNCaP lines additionally expressing luciferase (luc) in mice. (D) Raw images from the bioluminescence detection 29 days after tumor cell injection. (E) Luminescence values were plotted as an average of % of the first measurement (% relative bioluminescence) for each mouse in each respective group. The measurements were done in intact male mice at day 8, 15, 22, 29 and 41 after tumor cell injection. (F, G) Mice were surgically castrated after the 41 day-measurement and (F) luminescence was detected again after 6 weeks and (G) the % relative bioluminescence values were expressed in a box plot. The two groups consisted of 10 mice each. The animal weight between the two groups was assessed and did not significantly change. Statistical analysis was performed using a two-tailed student's t-test assuming unequal variance * p<0.05, ** p<0.01. Data are represented as mean +/- SEM.

Figure 7. The TRIM24 bromodomain and AR-interacting LxxLL motif are required for proliferation of CRPC cells

(A) Proliferation curves of abl cells stably expressing doxycycline (dox)-inducible shRNA targeting TRIM24 or a non-targeting (NT) control are shown. The shTRIM24 cell line was complemented with TRIM24 cDNA-rescue constructs resistant to shTRIM24, including TRIM24-WT (WT), TRIM24-L763A, L764A (L763A, L764A) and TRIM24-F979A, N980A (F979A, N980A). Cultures were treated with dox at day 0 and were grown and counted at 0, 3 and 6 days and values were plotted. Statistical analysis was performed using a two-tailed student's t-test assuming unequal variance ***p<0.005. (B) TRIM24 protein levels of the cell lines complemented with the indicated constructs were measured after 6 days of dox-treatment. (C, D) AR-specific ChIP followed by qPCR was performed at the AURKB enhancer, which is co-occupied by AR and TRIM24 (n=2). The different cell lines assayed are described in (A) and include (C) abl cells expressing shTRIM24 or shNT, and (D) abl shTRIM24 cell lines complemented with TRIM24-WT, TRIM24-L763A, L764A and TRIM24-F979A, N980A. All cell lines were treated with dox for 6 days to induce shRNA expression. The relative enrichment of the AR ChIP compared to the total input amount was set at 100% for the respective control condition. Statistical analysis was performed using a one-tailed student's t-test assuming equal variance * p<0.05, *** p<0.005. Data are represented as mean +/- SEM. (See also Figure S5).

Figure 8. Model of the oncogenic role of TRIM24 in CRPC

The functional role of TRIM24 is augmented in CRPC and depends on its interactions with AR (LxxLL motif) as well as acetylated histones (bromodomain) suggesting that. both TRIM24 functions could be targeted therapeutically