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Results: 9

1.
Figure 8

Figure 8. AR levels correlate inversely with RB levels and directly with E2F1 levels in CRPC specimens.. From: The retinoblastoma tumor suppressor controls androgen signaling and human prostate cancer progression.

(A) Log2 scaled expression ratios of AR versus RB1 and AR versus E2F1 from 39 human CRPC soft tissue metastases. (B) Heatmap depicting AR, RB1, E2F1, and E2F3 expression levels in 39 CRPC tumors. Pearson correlations are provided. See also Table 2 and Supplemental Figure 7.

Ankur Sharma, et al. J Clin Invest. 2010 December 1;120(12):4478-4492.
2.
Figure 9

Figure 9. Model for RB loss in controlling tumor progression through nuclear receptor expression and output.. From: The retinoblastoma tumor suppressor controls androgen signaling and human prostate cancer progression.

The present data suggest what we believe to be a new model for RB tumor suppressor function in tumor progression. While RB is actively engaged in response to hormone therapy, RB loss induces E2F1-mediated deregulation of the AR locus. This event is sufficient to induce unchecked AR activity and progression to CRPC. This model strongly suggests that RB plays a significant role in tumor progression that is independent of genes directly associated with cell cycle, manifest by controlling nuclear receptor expression, function, and output.

Ankur Sharma, et al. J Clin Invest. 2010 December 1;120(12):4478-4492.
3.
Figure 5

Figure 5. AR expression is induced in RB-deficient cells.. From: The retinoblastoma tumor suppressor controls androgen signaling and human prostate cancer progression.

(A) AR mRNA was monitored by qPCR, and AR protein levels by immunoblot, from cells cultured in the presence of androgen (FBS). For qPCR, expression in the shCon1 cohort was set to 1, and results represent triplicate analyses of at least 2–3 independent biological replicates (mean ± SD). *P < 0.05, Student’s t test. For immunoblots, Cdk4 served as a loading control. (B) Studies paralleling those of A were performed in L4-shCon1 and L4-shRB1 cells. (C) Intratumor AR mRNA levels monitored from xenograft studies described in Figure 3A. Immunoblots for intratumor AR protein levels are also shown. See also Supplemental Figure 5.

Ankur Sharma, et al. J Clin Invest. 2010 December 1;120(12):4478-4492.
4.
Figure 2

Figure 2. Loss of RB expression in CRPC is frequently associated with RB1 deletion. . From: The retinoblastoma tumor suppressor controls androgen signaling and human prostate cancer progression.

(A) RB1 mRNA expression, RB immunostaining, and RB1 copy number was concurrently measured using LuCaP xenografts derived from patients with CRPC. Representative images of RB immunostaining as a function of copy number are shown (original magnification, ×200). For samples retaining only a single RB1 locus, images for tumors found to harbor high and low RB1 mRNA expression are shown. LuCaP sample stained with control nonspecific anti–MOPC-21 antibody was used as a negative control. (B) Scatter plot for RB1 locus copy number versus average RB IHC score or RB1 transcript levels from the 22 LuCaP xenografts. *P < 0.05, ANOVA. (C) Average RB IHC immunostaining in 156 CRPC samples. Representative RB IHC images for tumor tissues with no RB staining (patient 05-116) and high RB staining (patient 00-090) are shown (original magnification, ×400).

Ankur Sharma, et al. J Clin Invest. 2010 December 1;120(12):4478-4492.
5.
Figure 6

Figure 6. The RB/E2F1 axis regulates the AR locus, and the kinetics of E2F1 and AR expression are cell cycle dependent. . From: The retinoblastoma tumor suppressor controls androgen signaling and human prostate cancer progression.

(A) Schematic of the AR regulatory locus, with the TSS and translational start site (TLS) shown. Results from ChIP analyses of E2F1 binding are shown, including semiquantitative and real-time PCR results, with occupancy in shCon1 cells set to 1. (B) ChIP analyses of RB binding, Sin3B, and histone H4 acetylation at sites enriched for E2F1 after RB depletion, and promoters of CCNA2 and ALB. (C) qPCR analyses of E2F1 and AR from LNCaP cells arrested in G0/G1 (CDT), G1 (ROS), early S-phase (APH), or mid–S-phase (HU). Transcript levels in CDT condition were set to 1. Data represent 3 replicates (mean ± SD); similar results were obtained in at least 3 independent analyses. See also Supplemental Figure 6.

Ankur Sharma, et al. J Clin Invest. 2010 December 1;120(12):4478-4492.
6.
Figure 7

Figure 7. Selective restoration of RB activity at sites of E2F1 action suppresses AR expression; AR regulation is E2F selective; and E2F1-induced AR deregulation is required for RB loss–mediated castration resistance.. From: The retinoblastoma tumor suppressor controls androgen signaling and human prostate cancer progression.

(A) Schematic of the E2F1-AB chimera. Also shown is RT-PCR for CCNA2 and AR mRNA from cells transfected with plasmids encoding E2F1, E2F1-AB, or control (pcDNA). (B) qPCR analyses of data in A, with AR levels in shCon1/control–transfected (pcDNA) cells set to 1. Data represent triplicate analyses of at least 2–3 independent biological replicates. (C) Immunoblot analyses for transduced E2F1, E2F2, E2F3, and lamin B (loading control) in LNCaP. (D) Parallel semiquantitative RT-PCR and qPCR analysis of AR mRNA in cells from C. Results are plotted relative to control (Ad-GFP–transduced LNCaP) from triplicate replicates (mean ± SD); similar results were obtained in at least 3 independent analyses. (E) AR immunoblot in control and E2F1-transduced cells from C and D. (F) Immunoblot for E2F1 and AR after siRNA-mediated knockdown of E2F1; verification of AR knockdown is also shown. Impact of E2F1 and AR knockdown on castration-resistant proliferation in RB-depleted cells was determined by trypan blue exclusion and cell counting. Data reflect duplicate experiments, each with 3 independent biologic replicates. *P < 0.05, Student’s t test.

Ankur Sharma, et al. J Clin Invest. 2010 December 1;120(12):4478-4492.
7.
Figure 4

Figure 4. RB loss deregulates AR occupancy at target gene loci and E2F1 expression under therapeutic conditions.. From: The retinoblastoma tumor suppressor controls androgen signaling and human prostate cancer progression.

(A) PSA locus depicting AREs relative to the TSS is illustrated. AR occupancy and output was determined at the indicated time points after 10 nM DHT stimulation; for comparison, AR occupancy in shCon1 at 0 hours was set to 1. In parallel, PSA mRNA was quantified relative to 18S control, and expression in shCon1 at the 0-hour time point was set to 1. (B) AR occupancy in both cell types was determined using loci specific to CRPC. (C) AR occupancy was determined as in A, but after stimulation with 10 μM Bic. (D) E2F1 expression was quantified by qPCR and immunoblot in LNCaP and LAPC4 cells after RB knockdown. qPCR data in AD represent mean ± SD of 3 replicates; similar results were obtained in at least 2 independent experiments. (E) Immunoblots for E2F1 after 48 hours of androgen ablation or under parallel conditions supplemented with 1 μM Bic. (F) Immunoblot of E2F1 from representative tumors described in Figure 3 at sacrifice. See also Supplemental Figures 3 and 4.

Ankur Sharma, et al. J Clin Invest. 2010 December 1;120(12):4478-4492.
8.
Figure 1

Figure 1. RB loss is overrepresented in CRPC and metastatic PCa and is associated with tumor recurrence.. From: The retinoblastoma tumor suppressor controls androgen signaling and human prostate cancer progression.

(A) RB expression was determined in non-neoplastic tissue, localized PCa, and CRPC from an established dataset (54) plotted as log of robust multichip average (RMA) expression. For all box plots, the red line is the median, the bottom and top of the box are the 25th and 75th percentiles, respectively, and the whiskers capture data points within 1.5 interquartile range (IQR). Outliers beyond 1.5 IQR are plotted individually with gray plus symbols. (B) The RB loss signature was determined from data in A, and plotted as a box and whisker plot and heatmap. In the heatmap, tissue samples were ordered from left to right based on relative representation of the RB loss signature (above). Tissue type is provided below. (C) RB loss signature in primary PCa and metastatic PCa, from data first described elsewhere (55). Also shown is RB loss signature across normal, benign prostate epithelia (BPH), localized PCa, and metastatic specimens. As in B, tissue samples were ordered from left to right based on relative level of the RB loss signature, and sample type is provided below. Indicated P values were calculated using the Student’s t test. (D) Quartile analyses were used to determine the impact of RB loss on recurrence-free survival using the dataset in C. See also Supplemental Figure 8.

Ankur Sharma, et al. J Clin Invest. 2010 December 1;120(12):4478-4492.
9.
Figure 3

Figure 3. RB depletion enables bypass of hormonal therapy.. From: The retinoblastoma tumor suppressor controls androgen signaling and human prostate cancer progression.

(A) Immunoblot for RB in shCon1 and shRB1 cells. Relative tumor volume of xenografts after castration is also shown. Hosts were castrated when tumors reached 100–150 mm3 (day 0). Data plotted are mean tumor size ± SD for each cohort. n = 10 (shCon1); 7 (shRB1). (B) Tumor mass at sacrifice (day 28). Individual data points represent the tumor mass of each xenograft at sacrifice, and the mean for each cohort is represented by a horizontal bar. (C) Relative serum PSA for 4 weeks after castration, beginning at the nadir (day 7 after castration, set to 1). (D) Serum PSA doubling time for each cohort was determined as follows: time (days) × loge (2)/[loge (PSA28) – loge (PSA7)], where PSA7 and PSA28 represent PSA levels at days 7 and 28 after castration. n = 9 (shCon1); 7 (shRB1). (E) Intratumor PSA mRNA levels in xenograft tissues at sacrifice were determined via qPCR. PSA relative to 18S is plotted; expression in shCon1 was set to 1. n = 5 (shCon1); 7 (shRB1). (F) PSA and TMPRSS2 mRNA levels were determined by qPCR in cells cultured for 48 hours in androgen-free (CDT) or androgen-containing (FBS) media and supplemented as indicated. Results are plotted for each treatment condition relative to that in shCon1 cells after androgen ablation (set to 1). Data reflect triplicate analyses of at least 2–3 independent biological replicates (mean ± SD). *P < 0.05, Student’s t test. See also Supplemental Figures 1 and 2.

Ankur Sharma, et al. J Clin Invest. 2010 December 1;120(12):4478-4492.

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