Research ArticleCancer

Cell type–specific abundance of 4EBP1 primes prostate cancer sensitivity or resistance to PI3K pathway inhibitors

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Science Signaling  17 Nov 2015:
Vol. 8, Issue 403, pp. ra116
DOI: 10.1126/scisignal.aad5111
  • Fig. 1 Luminal epithelial prostate cancer cells are enriched upon ATP site inhibition of mTOR and are characterized by increased 4EBP1 protein abundance and lower baseline protein synthesis rates in vivo.

    (A) Schematic of MLN0128 preclinical trial. Age-matched 9- to 12-month-old PTENL/L mice were treated with vehicle or MLN0128 (1 mg/kg) daily for 8 weeks (upper panel). Quantification for ratio of CK8+ luminal epithelial cells over CK5+ basal epithelial cells in vehicle- or MLN0128-treated PTENL/L mice (lower panel; n = 6 mice per condition; *P < 0.05, t test). (B) Representative Western blots for 4EBP1, eIF4A, eIF4E, eIF4G, phospho-4EBP1 (Thr37/46), phospho–rpS6 (ribosomal protein S6) (Ser240/244), and rpS6 in basal epithelial and luminal epithelial cells from PTENL/L mice treated with and without MLN0128 (1 mg/kg) daily for 4 days by oral gavage. (C) Representative Western blot for 4EBP1 in basal epithelial and luminal epithelial cells from wild-type (WT) mice (left panel). Quantification of 4EBP1 densitometry in both WT and PTENL/L basal and luminal epithelial cells (right panel; n = 3 mice per genotype; *P = 0.04, t test). (D) 4EBP1 mRNA expression in WT and PTENL/L basal and luminal epithelial cells (n = 2 to 3 mice per genotype; *P = 0.002, t test). All samples were first normalized to actin. (E) Representative [35S]methionine incorporation run on the same gel in sorted basal and luminal epithelial cell types from WT and PTENL/L mice (left panel). Quantification of [35S]methionine incorporation by densitometry in sorted basal and luminal epithelial cell types from WT and PTENL/L mice (right panel; n = 3 mice per genotype; *P < 0.0001, **P = 0.002, t test). Data are means ± SEM. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; n.s., not significant.

  • Fig. 2 Inhibition of eIF4E activity does not affect WT prostate epithelial cell maintenance but suppresses prostate tumor initiation and progression in the setting of PTEN loss in vivo.

    (A) Schematic representation of the prostate-specific and doxycycline-inducible PTENL/L;4EBP1M mouse model, in which the addition of doxycycline (dox) to the drinking water (at 2 g/liter) induced the expression of the 4EBP1M transgene. (B) Representative hematoxylin and eosin (H&E) staining of ventral prostate glands from 4EBP1M mice after 4 weeks with or without doxycycline in their drinking water. Scale bar, 100 μm. (C) Representative H&E staining of WT, PTENL/L, and PTENL/L;4EBP1M prostates after 4 to 5 weeks of exposure to doxycycline after weaning. Percent high-grade prostatic intraepithelial neoplasia (PIN)–positive glands in PTENL/L and PTENL/L;4EBP1M mice (n = 3 mice per genotype, *P = 0.03, t test). Scale bar, 500 μm. (D) Fold change in TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling)–positive cells in WT, PTENL/L, and PTENL/L;4EBP1M prostates after 4 to 5 weeks of exposure to doxycycline after weaning (n = 3 mice per genotype; *P = 0.004, **P < 0.0001, t test). (E) Representative PTENL/L;4EBP1M prostate with or without exposure to doxycycline for 8 weeks starting at age 6 to 8 months. Mice were at 8 to 10 months of age at necropsy. (F) Quantification of mouse prostate weights between PTENL/L and PTENL/L;4EBP1M exposed to doxycycline for 8 weeks (n = 4 to 5 mice per genotype; *P = 0.02, t test). (G) Representative ultrasounds of PTENL/L and PTENL/L;4EBP1M anterior prostates before and after 8 weeks of exposure to doxycycline. (H) Quantification of tumor area in PTENL/L and PTENL/L;4EBP1M anterior prostates before and after 8 weeks of exposure to doxycycline (n = 4 to 5 mice per genotype; *P = 0.003, t test). (I) Fold change in TUNEL-positive (apoptotic) cells in PTENL/L and PTENL/L;4EBP1M (n = 3 mice per genotype; *P = 0.0006, t test). Data are means ± SEM.

  • Fig. 3 PTENL/L luminal epithelial cells are enriched upon expression of 4EBP1M.

    (A) Fold change in basal and luminal epithelial cell numbers upon PTEN loss in WT and PTENL/L mice (n = 7 to 9 mice per genotype; *P = 0.005, **P = 0.01, t test). (B) Ratio of CK8+ luminal epithelial cells over CK5+ basal epithelial cells in PTENL/L and PTENL/L;4EBP1M mice (n = 6 mice per condition; *P = 0.001, t test). (C) Percent 5-bromo-2′-deoxyuridine (BrdU) incorporation by FACS of the basal and luminal epithelial cell types from mice exposed to doxycycline (administered in the drinking water at 2 g/liter) at weaning for a total of 4 to 5 weeks. Upper: Representative BrdU dot plot. Lower: Quantification of percent BrdU-positive basal and luminal cells in each mouse strain (n = 5 to 6 mice per genotype, t test). FSC, forward scatter. (D) 7AAD/annexin FACS for apoptosis in basal and luminal epithelial cell types from mice exposed to the doxycycline regimen upon weaning for a total of 4 to 5 weeks. Upper: Representative 7AAD/annexin zebra plot. Lower: Quantification of 7AAD/annexin double-positive cells in basal or luminal cells in each mouse strain (n = 6 to 7 mice per genotype; *P = 0.03, t test). All mice were at 8 to 10 weeks of age at necropsy. Data are means ± SEM.

  • Fig. 4 Increased abundance of 4EBP1 is required to maintain resistance to PI3K pathway inhibitors and is a marker of resistant cells in human prostate cancer.

    (A) Representative Western blot analysis from two experiments for PTEN and 4EBP1 in PTEN KD LHS PrEC clones and control LHS cells. (B) Analysis of apoptosis by propidium iodide/annexin V staining in PTEN KD LHS PrECs after 12-hour exposure to MLN0128 or vehicle (–MLN0128) (n = 5 replicates in two independent experiments; *P < 0.0001, **P = 0.04, t test). (C) Representative Western blot of 4EBP1 in PTEN KD LHS PrECs with and without si4EBP1. (D) Analysis of apoptosis by propidium iodide/annexin V staining of PTEN KD LHS PrECs after transfection with a 4EBP1-targeted siRNA pool (n = 8 replicates in two independent experiments; *P < 0.0001, **P = 0.005, t test). (E) Representative autoradiograph (left) and quantification of [35S]methionine incorporation assays in PTEN KD LHS PrEC clones upon silencing 4EBP1 [n = 3 independent experiments; *P < 0.05, ANOVA (analysis of variance)]. (F) Representative cap-binding assay in PTEN KD LHS clones upon silencing 4EBP1. Data are means ± SEM.

  • Fig. 5 Increased 4EBP1 abundance is associated with drug resistance in prostate cancer patients.

    (A) Schematic of the phase 2 neoadjuvant BKM120 clinical trial conducted at the University of California, San Francisco (UCSF). (B) Representative H&E, CK8, and 4EBP1 immunofluorescence images of a patient tumor before and after treatment with the PI3K inhibitor BKM120. Far right: Magnified insets from the 4EBP1 images. Scale bars, 50 and 10 μm, respectively. (C) Quantification of the percentage of luminal epithelial cells with low or high abundance of 4EBP1 for each patient as well as the average for all of the patients before (pre) and after (post) treatment with BKM120 (*P < 0.0001, t test).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/403/ra116/DC1

    Fig. S1. Absolute quantification of basal epithelial cells and luminal epithelial cells after treatment with MLN0128.

    Fig. S2. qPCR phenotyping of distinct sorted epithelial cell populations in wild-type and PTENL/L mice.

    Fig. S3. Efficiency of PTEN deletion and the phosphorylation and localization of 4EBP1 in basal and luminal epithelial cells in vivo.

    Fig. S4. Western blot analysis of translation initiation factors and regulators in wild-type and PTENL/L basal and luminal epithelial cells.

    Fig. S5. 4EBP1M does not affect normal prostate homeostasis but impedes prostate cancer initiation and progression.

    Fig. S6. Effect of PTEN loss and 4EBP1M expression on absolute number of basal and luminal epithelial cells.

    Fig. S7. Expression of 4EBP1 endows resistance to MLN0128 in LHS PTEN KD cell lines.

    Fig. S8. Phosphorylated AKT (Ser473) immunohistochemistry of prostate tumors and serum PSA concentrations from patients before and after treatment with BKM120.

    Table S1. qPCR oligonucleotide sequences.

  • Supplementary Materials for:

    Cell type–specific abundance of 4EBP1 primes prostate cancer sensitivity or resistance to PI3K pathway inhibitors

    Andrew C. Hsieh,* Hao G. Nguyen, Lexiaochuan Wen, Merritt P. Edlind, Peter R. Carroll, Won Kim, Davide Ruggero*

    *Corresponding author. E-mail: ahsieh{at}fredhutch.org (A.C.H.); davide.ruggero{at}ucsf.edu (D.R.)

    This PDF file includes:

    • Fig. S1. Absolute quantification of basal epithelial cells and luminal epithelial cells after treatment with MLN0128.
    • Fig. S2. qPCR phenotyping of distinct sorted epithelial cell populations in wild-type and PTENL/L mice.
    • Fig. S3. Efficiency of PTEN deletion and the phosphorylation and localization of 4EBP1 in basal and luminal epithelial cells in vivo.
    • Fig. S4. Western blot analysis of translation initiation factors and regulators in wild-type and PTENL/L basal and luminal epithelial cells.
    • Fig. S5. 4EBP1M does not affect normal prostate homeostasis but impedes prostate cancer initiation and progression.
    • Fig. S6. Effect of PTEN loss and 4EBP1M expression on absolute number of basal and luminal epithelial cells.
    • Fig. S7. Expression of 4EBP1 endows resistance to MLN0128 in LHS PTEN KD cell lines.
    • Fig. S8. Phosphorylated AKT (Ser473) immunohistochemistry of prostate tumors and serum PSA concentrations from patients before and after treatment with BKM120.
    • Table S1. qPCR oligonucleotide sequences.

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    Citation: A. C. Hsieh, H. G. Nguyen, L. Wen, M. P. Edlind, P. R. Carroll, W. Kim, D. Ruggero, Cell type–specific abundance of 4EBP1 primes prostate cancer sensitivity or resistance to PI3K pathway inhibitors. Sci. Signal. 8, ra116 (2015).

    © 2015 American Association for the Advancement of Science

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