Research ArticleCancer

The androgen receptor regulates a druggable translational regulon in advanced prostate cancer

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Science Translational Medicine  31 Jul 2019:
Vol. 11, Issue 503, eaaw4993
DOI: 10.1126/scitranslmed.aaw4993
  • Fig. 1 AR controls translation initiation via a cis-element encoded within the 4ebp1 locus.

    (A) Representative puromycin immunofluorescence for de novo protein synthesis in vivo in intact and 8-week castrate PtenL/L ventral prostates (left). Violin plot of per cell quantitation of puromycin mean fluorescence intensity. The height of the plot represents the range of new protein synthesis observed, and the width represents the number of cells at each fluorescence intensity [right; intact, n = 3 (46,711 cells quantified); castrate, n = 4 (73,237 cells quantified); *P < 2.2 × 10−16, t test]. DAPI, 4′,6-diamidino-2-phenylindole. (B) Simplified schematic of the eIF4F translation initiation complex composed of eIF4E, eIF4G, and eIF4A with the inhibitor of the complex, 4EBP1 (P, phosphorylation; AUG, start codon). (C) Representative immunofluorescence for eIF4E, eIF4G, eIF4A, and 4EBP1 in intact and 8-week castrate PtenL/L ventral prostates (left). Violin plot of per cell quantitation of 4EBP1 mean fluorescence intensity [right; intact, n = 6 (148,974 cells quantified); castrate, n = 5 (111,046 cells quantified); *P < 2.2 × 10−16, t test]. (D) Representative Western blot for AR, 4EBP1, and actin in human AR+ parental and AR APIPC (AR program-independent prostate cancer) cells. (E) Correlation plot of 29 human nonneuroendocrine CRPC LuCaP prostate cancer PDX models comparing AR protein content (y axis, AR H score) and 4EBP1 protein expression [x axis, 4EBP1 mean fluorescence intensity (MFI)] (R = 0.376, P = 0.02, Spearman’s correlation). (F) 4ebp1 mRNA expression by RNA-seq in intact and 8-week castrate PtenL/L ventral prostates (intact, n = 2; castrate, n = 3; *P = 0.002, t test). (G) 4ebp1 mRNA expression by qPCR in primary intact (DHT+) and castrate (DHT) PtenL/L prostate cancer cells. DHT (1 nM) was added back to castrate cells over the indicated time points (three biological replicates; *P < 0.05, t test). (H) Schematic of the wild-type (WT) and mutant 4ebp1 intron reporter constructs cloned into the pGL4.28 vector (red triangle, minimal promoter region; Luc, firefly luciferase). Representative Western blot of AR upon addition of testosterone analog dimethylnortestosterone (DMNT) in LNCaP cells (left). Luciferase assay of the putative wild-type and mutated (MUT) mouse 4ebp1 androgen response element (mARE) [right; six biological replicates; *P < 0.0001, analysis of variance (ANOVA)]. Scale bars, 100 μm. Data are presented as means ± SEM.

  • Fig. 2 4EBP1 expression controls eIF4E-eIF4G interaction dynamics and proliferation in a cell-autonomous manner.

    (A) Schematic of the eIF4E-eIF4G and eIF4E-4EBP1 proximity ligation assays, which allow for the quantification of eIF4F translation initiation complexes and 4EBP1 inhibitory complexes in vivo. (B) Representative images of the eIF4E-eIF4G and eIF4E-4EBP1 proximity ligation assays in intact and 8-week castrate PtenL/L ventral prostates (left). Quantification of the proximity ligation assay (right; intact, n = 6; castrate, n = 7; *P = 0.03 and **P = 0.009, t test). (C) Representative hematoxylin and eosin staining of intact and 8-week castrate PtenL/L ventral prostates (left) with quantification (right; intact, n = 8; castrate, n = 10; *P = 0.04, t test). (D) Representative Ki67 staining of intact and 8-week castrate PtenL/L ventral prostates (left panel) with quantification [right panel: intact, n = 7 (151 glands quantified); castrate, n = 9 (206 glands quantified); *P < 0.0001, t test]. (E) Representative Western blot for PTEN and actin in wild-type, intact PtenL/L, and 8-week castrate PtenL/L primary organoids (top). Representative Western blot for AR, 4EBP1, and actin in intact PtenL/L and 8-week castrate PtenL/L primary organoids (bottom panel). (F) Growth curves of intact and castrate PtenL/L primary cells (three biological replicates; P = 0.03, t test). Scale bars, 100 μm. Data are presented as means ± SEM.

  • Fig. 3 AR and eIF4F-mediated mRNA-specific translation controls a regulon of functional cell proliferation regulators.

    (A) Probability density graph of 697 translationally up-regulated mRNAs between intact (n = 2) and castrate (n = 3) PtenL/L ventral prostates. Translation efficiency, ribosome-bound mRNA/total mRNA (P < 2.2 × 10−16, Kolmogorov-Smirnov test). (B) Folding energy (*P = 0.004339) and %GC content (*P < 2.2 × 10−16) between 5′UTRs of control mRNA (n = 19,009) and up-regulated mRNA (n = 187, t test). Whiskers represent 1.5 times the interquartile range. (C) The GRTE consensus sequence (e = 1.2× 10−41). (D) Luciferase assay of the control vector, wild-type Klf5 5′UTR luciferase construct, and its GRTE deletion mutant with or without 4EBP1M induction. Luciferase assay was normalized to luc and RPS19 mRNA (n.s., not statistically significant; n > 3 biological replicates per condition, t test). (E) Gene set enrichment analysis of the translationally up-regulated mRNA (log2 fold change, ≥0.75; FDR < 0.1) in castrate PtenL/L mice. (F) Heatmap of translationally up-regulated proliferation regulators in AR-low prostate cancer (log2 fold change, ≥0.75; FDR < 0.1). (G) Representative Western blot analysis of KLF5, DENR, CACUL1, rpS15, AR, and actin in primary intact (In; DHT+) and castrate (Cx; DHT) PtenL/L organoids. (H) Representative Western blot analysis of KLF5, DENR, CACUL1, rpS15, AR, and actin in primary PtenL/L;4ebp1M organoids with or without 4EBP1M induction. (I) Cell proliferation EdU incorporation assay in scramble, shKLF5, shDENR, or shCACUL castrate (DHT) PtenL/L primary cells (replicate of four to six per condition; *P = 0.02, **P < 0.0001, and ***P = 0.0003, t test). Data are presented as means ± SEM.

  • Fig. 4 Increased eIF4F complex formation is necessary for AR-low prostate cancer initiation and progression.

    (A) Schematic diagram of testing the impact of inhibiting eIF4F complex formation on AR-low prostate cancer initiation. PtenL/L;4ebp1M mice were castrated and immediately put on vehicle or doxycycline (dox) for 8 weeks. (B) Representative hematoxylin and eosin staining of vehicle-treated (−4EBP1M) and doxycycline-treated (+4EBP1M) PtenL/L;4ebp1M ventral prostates (left). Quantification of tumor volumes after 8 weeks of inhibition of eIF4F complex formation started immediately after castration (right; vehicle, n = 9; doxycycline, n = 9; *P = 0.04). (C) Representative Ki67 staining of vehicle-treated (−4EBP1M) and doxycycline-treated (+4EBP1M) PtenL/L;4ebp1M ventral prostates (left). Ki67 quantification after 8-week castration and concurrent vehicle or doxycycline treatment [right; vehicle, n = 9 (205 glands quantified); doxycycline, n = 8 (169 glands quantified); *P < 0.0001, t test]. (D) Schematic diagram of testing the impact of inhibiting eIF4F assembly on AR-low prostate cancer progression. PtenL/L;4ebp1M mice were castrated and allowed to form AR-low tumors for 12 weeks followed by an additional 12-week vehicle or doxycycline treatment. (E) PtenL/L;4ebp1M ventral prostate weights after a 12-week castration followed by an additional 12-week vehicle or doxycycline treatment (vehicle, n = 10; doxycycline, n = 9; *P = 0.0018, t test). (F) Representative images of PtenL/L;4ebp1M ventral prostates with or without 4ebp1M induction in the progression experiment. (G) PtenL/L;4ebp1M ventral prostate Ki67 quantification after a 12-week castration followed by an additional 12-week vehicle or doxycycline treatment [vehicle, n = 9 (197 glands quantified); doxycycline, n = 7 (139 glands quantified); *P < 0.0001, t test]. Scale bars, 100 μm. Data are presented as means ± SEM.

  • Fig. 5 AR-low prostate cancer is more sensitive to disruption of the eIF4E-eIF4G interaction than AR-intact prostate cancer.

    (A) Intact and castrate PtenL/L;4ebp1M primary prostate cancer cells treated with doxycycline for 48 hours. Proliferation was measured using the IncuCyte platform (assay completed in triplicate; *P = 0.0026 and **P = 0.03, t test). (B) 4EBP1 protein immunofluorescence quantification of a tissue microarray composed of end-stage metastatic CRPC patient specimens classified by AR protein expression (two to four tumors sampled per patient; AR low, n = 10; AR high, n = 17; *P = 0.0089, t test). (C) Simplified schematic of the mechanism of action of 4E1RCat, 4E2RCat, and 4EGI-1, which disrupt the eIF4E-eIF4G interaction. (D) Intact and castrate PtenL/L cells treated with 4E2RCat for 48 hours. Proliferation was measured using the IncuCyte platform (assay completed in triplicate; *P < 0.0001, t test). (E) Intact and castrate PtenL/L cells treated with 4EGI-1 for 48 hours. Proliferation was measured using the IncuCyte platform (assay completed in triplicate; *P = 0.002, t test). (F) AR+ parental and AR APIPC prostate cancer cells treated with 4E2RCat for 48 hours. Proliferation was measured using the IncuCyte platform (assay completed in triplicate; *P < 0.0001 and **P = 0.0003, t test). (G) AR+ parental and AR APIPC prostate cancer cells treated with 4EGI-1 for 48 hours. Proliferation was measured using the IncuCyte platform (assay completed in triplicate; *P = 0.0003, t test). Data are presented as means ± SEM.

  • Fig. 6 Targeting the eIF4E-eIF4G interaction in AR-deficient prostate cancer decreases tumor growth and improves survival.

    (A) Schematic of the eIF4E-eIF4G interaction inhibitor preclinical trials. (B) AR APIPC xenograft preclinical trial testing the efficacy of 4E1RCat on AR-low prostate cancer tumor growth. Castrated mice were treated with 4E1RCat or vehicle (15 mg/kg; 4E1RCat-treated, n = 8; vehicle-treated mice, n = 7; *P = 0.0124, **P = 0.045, and ***P = 0.05, t test). (C) AR APIPC xenograft preclinical trial testing the impact of 4E1RCat on AR-low prostate cancer survival. Castrated mice were treated with 4E1RCat or vehicle (15 mg/kg; 4E1RCat-treated, n = 8; vehicle-treated mice, n = 7; P = 0.0048, log-rank test). (D) LuCaP 173.2 PDX preclinical trial testing the efficacy of 4E1RCat on AR-low prostate tumor growth. Castrated mice were treated with 4E1RCat or vehicle (15 mg/kg; 4E1RCat-treated, n = 9; vehicle-treated mice, n = 8; *P = 0.02 and **P = 0.01, t test). (E) LuCaP 173.2 PDX preclinical trial testing the impact of 4E1RCat in AR-low prostate cancer survival. Castrated mice were treated with 4E1RCat or vehicle (15 mg/kg; 4E1RCat-treated, n = 9; vehicle-treated mice, n = 8; P = 0.0057, log-rank test). (F) AR+ parental APIPC xenograft preclinical trial testing the efficacy of 4E1RCat on AR+ prostate cancer tumor growth. Uncastrated mice were treated with 4E1RCat or vehicle (15 mg/kg; 4E1RCat-treated, n = 8; vehicle-treated mice, n = 7). Data are presented as means ± SEM.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/503/eaaw4993/DC1

    Materials and Methods

    Fig. S1. Castration of PtenL/L mice decreases AR, AR activity, and 4EBP1 without affecting eIF4F components.

    Fig. S2. AR regulates 4ebp1 transcription but does not affect translation efficiency or degradation rates.

    Fig. S3. Androgen deprivation is associated with decreased 4EBP1 expression; DHT add back decreases de novo protein synthesis.

    Fig. S4. AR binds to an ARE in 4ebp1 in both normal and cancerous prostates, rendering 4EBP1 AR responsive.

    Fig. S5. Castrate PtenL/L mice develop highly aggressive, nonneuroendocrine tumors independent of PI3K or MNK1/2 activity.

    Fig. S6. AR/eIF4F-sensitive mRNAs are distinct from mTOR inhibition–sensitive mRNAs.

    Fig. S7. Protein but not mRNA expression of GRTE-containing proliferation regulators is responsive to changes in eIF4F activity.

    Fig. S8. Decreased eIF4F complex formation by 4EBP1M results in smaller and less aggressive tumors in castrate PtenL/L;4ebp1M mice.

    Fig. S9. Castrate PtenL/L mice exhibit increased sensitivity to eIF4F disruption; 4EBP1 abundance is independent of AR in HSPC.

    Fig. S10. 4E2RCat and 4EGI-1 disrupt eIF4F complex formation in PtenL/L cells, AR+ parental, and AR APIPC cells.

    Fig. S11. AR- and eIF4F-targeted combinatorial treatments in LNCaP prostate cancer cells demonstrate antitumor activity.

    Fig. S12. AR shapes the prostate cancer proteome through 4EBP1 and a druggable pro-proliferation translational regulon.

    Table S1. mRNA expression of AR signature genes comparing castrate PtenL/L ventral prostates to intact PtenL/L ventral prostates.

    Table S2. Position-weighted map of the 5′UTR GRTE.

    Table S3. Primers used in this study.

    Data file S1. Translationally up-regulated genes in the castrate PtenL/L mouse.

    Data file S2. Tumor measurements from in vivo experiments.

    References (4547)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Castration of PtenL/L mice decreases AR, AR activity, and 4EBP1 without affecting eIF4F components.
    • Fig. S2. AR regulates 4ebp1 transcription but does not affect translation efficiency or degradation rates.
    • Fig. S3. Androgen deprivation is associated with decreased 4EBP1 expression; DHT add back decreases de novo protein synthesis.
    • Fig. S4. AR binds to an ARE in 4ebp1 in both normal and cancerous prostates, rendering 4EBP1 AR responsive.
    • Fig. S5. Castrate PtenL/L mice develop highly aggressive, nonneuroendocrine tumors independent of PI3K or MNK1/2 activity.
    • Fig. S6. AR/eIF4F-sensitive mRNAs are distinct from mTOR inhibition–sensitive mRNAs.
    • Fig. S7. Protein but not mRNA expression of GRTE-containing proliferation regulators is responsive to changes in eIF4F activity.
    • Fig. S8. Decreased eIF4F complex formation by 4EBP1M results in smaller and less aggressive tumors in castrate PtenL/L;4ebp1M mice.
    • Fig. S9. Castrate PtenL/L mice exhibit increased sensitivity to eIF4F disruption; 4EBP1 abundance is independent of AR in HSPC.
    • Fig. S10. 4E2RCat and 4EGI-1 disrupt eIF4F complex formation in PtenL/L cells, AR+ parental, and AR APIPC cells.
    • Fig. S11. AR- and eIF4F-targeted combinatorial treatments in LNCaP prostate cancer cells demonstrate antitumor activity.
    • Fig. S12. AR shapes the prostate cancer proteome through 4EBP1 and a druggable pro-proliferation translational regulon.
    • Table S1. mRNA expression of AR signature genes comparing castrate PtenL/L ventral prostates to intact PtenL/L ventral prostates.
    • Table S2. Position-weighted map of the 5′UTR GRTE.
    • Table S3. Primers used in this study.
    • References (4547)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Translationally up-regulated genes in the castrate PtenL/L mouse.
    • Data file S2 (Microsoft Excel format). Tumor measurements from in vivo experiments.

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