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

Abstract

Pharmacological inhibitors against the PI3K-AKT-mTOR (phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin) pathway, a frequently deregulated signaling pathway in cancer, are clinically promising, but the development of drug resistance is a major limitation. We found that 4EBP1, the central inhibitor of cap-dependent translation, was a critical regulator of both prostate cancer initiation and maintenance downstream of mTOR signaling in a genetic mouse model. 4EBP1 abundance was distinctly different between the epithelial cell types of the normal prostate. Of tumor-prone prostate epithelial cell types, luminal epithelial cells exhibited the highest transcript and protein abundance of 4EBP1 and the lowest protein synthesis rates, which mediated resistance to both pharmacologic and genetic inhibition of the PI3K-AKT-mTOR signaling pathway. Decreasing total 4EBP1 abundance reversed resistance in drug-insensitive cells. Increased 4EBP1 abundance was a common feature in prostate cancer patients who had been treated with the PI3K pathway inhibitor BKM120; thus, 4EBP1 may be associated with drug resistance in human tumors. Our findings reveal a molecular program controlling cell type-specific 4EBP1 abundance coupled to the regulation of global protein synthesis rates that renders each epithelial cell type of the prostate uniquely sensitive or resistant to inhibitors of the PI3K-AKT-mTOR signaling pathway.

Figure 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 9–12 month-old PTEN^L/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 PTEN^L/L mice (lower panel, n = 6 mice/condition, *P<0.05, t-test). (B) Representative Western blots for 4EBP1, eIF4A, eIF4E, eIF4G, phospho-4EBP1 (Thr^37/46), phospho-rpS6 (Ser^240/244), and rpS6 in basal epithelial and luminal epithelial cells from PTEN^L/L mice treated with and without MLN0128 1mg/kg daily for 4 days by oral gavage. (C) Representative Western blot for 4EBP1 in basal epithelial and luminal epithelial cells from WT mice (left panel). Quantification of 4EBP1 densitometry in both wild-type (WT) and PTEN^L/L basal and luminal epithelial cells (right panel, n = 3 mice/genotype, *P = 0.04, t-test). (D) 4EBP1 mRNA expression in WT and PTEN^L/L basal and luminal epithelial cells (n = 2–3 mice/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 PTEN^L/L mice (left panel). Quantification of [³⁵S]-methionine incorporation by densitometry in sorted basal and luminal epithelial cell types from WT and PTEN^L/L mice (right panel, n = 3 mice/genotype, *P<0.0001, **P = 0.002, t-test). Data are mean +/− S.E.M.

Figure 2. Inhibition of eIF4E activity does not affect wild-type 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 PTEN^L/L;4EBP1^M mouse model, in which the addition of doxycycline to the drinking water (at 2 g/L) induced the expression of the 4EBP1^M transgene. (B) Representative H+E staining of ventral prostate glands from 4EBP1^M mice after 4 weeks with or without doxycycline in their drinking water. Scale bar, 100 μm. (C) Representative H+E staining of wild-type (WT), PTEN^L/L, and PTEN^L/L;4EBP1^M prostates after 4–5 weeks of exposure to doxycycline after weaning. Percent high grade prostatic intraepithelial neoplasia (PIN) positive glands in PTEN^L/L and PTEN^L/L;4EBP1^M mice (n = 3 mice/genotype, *P = 0.03, t-test). Scale bar, 500 μm. (D) Fold change in TUNEL positive cells in WT, PTEN^L/L, and PTEN^L/L;4EBP1^M prostates after 4–5 weeks of exposure to doxycycline after weaning (n = 3 mice/genotype, *P = 0.004, **P <0.0001, t-test). (E) Representative PTEN^L/L;4EBP1^M prostate with or without exposure to doxycycline for 8 weeks starting at age 6–8 months. Mice were at 8–10 months of age at necropsy. (F) Quantification of mouse prostate weights between PTEN^L/L and PTEN^L/L;4EBP1^M exposed to doxycycline for 8 weeks (n = 4–5 mice/genotype, *P = 0.02, t-test). (G) Representative ultrasounds of PTEN^L/L and PTEN^L/L;4EBP1^M anterior prostates before and after 8 weeks exposure to doxycycline. (H) Quantification of tumor area in PTEN^L/L and PTEN^L/L;4EBP1^M anterior prostates before and after 8 weeks exposure to doxycycline (n = 4–5 mice / genotype, *P= 0.003, t-test). (I) Fold change in TUNEL-positive (apoptotic) cells in PTEN^L/L and PTEN^L/L;4EBP1^M (n = 3 mice/genotype, *P = 0.0006, t-test). Data are mean +/− S.E.M.

Figure 3. PTEN^L/L luminal epithelial cells are enriched upon expression of 4EBP1^M

(A) Fold change in basal and luminal epithelial cell numbers upon PTEN loss in wild-type (WT) and PTEN^L/L mice (n = 7–9 mice/genotype, *P = 0.005, **P = 0.01, t-test). (B) The ratio of CK8+ luminal epithelial cells over CK5+ basal epithelial cells in PTEN^L/L and PTEN^L/L;4EBP1^M mice (n = 6 mice/condition,*P = 0.001, t-test). (C) Percent BrdU incorporation by fluorescence-activated cell sorting (FACS) of the basal and luminal epithelial cell types from mice exposed to doxycycline (administered in the drinking water at 2 g/L) at weaning for a total of 4–5 weeks. Upper panel: representative BrdU dot plot. Lower panel: quantification of percent BrdU positive basal and luminal cells in each mouse strain. FSC, forward scatter. (n = 5–6 mice/genotype, t-test). (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 4–5 weeks. Upper panel: representative 7AAD/Annexin dot plot. Lower panel: quantification of 7AAD/Annexin double positive cells in basal or luminal cells in each mouse strain (n = 6–7 mice/genotype, *P = 0.03, t-test). All mice were at 8–10 weeks of age at necropsy. n.s. = not statistically significant. Data are mean +/− S.E.M.

Figure 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 2 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 PrEC cells after 12 hours’ 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 PrEC cells with and without si4EBP1. (D) Analysis of apoptosis by propidium iodide/annexin V staining of PTEN-KD LHS PrEC cells 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 S³⁵-methionine incorporation assays in PTEN KD LHS PrEC clones upon silencing 4EBP1. (n = 3 independent experiments, *P < 0.05, ANOVA). (F) Representative cap-binding assay in PTEN knockdown LHS clones upon silencing 4EBP1. n.s. = not statistically significant. Data are mean ± S.E.M.

Figure 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 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 μm 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).