The androgen receptor is a negative regulator of eIF4E phosphorylation at S209: implications for the use of mTOR inhibitors in advanced prostate cancer

Abstract

The antiandrogen bicalutamide is widely used in the treatment of advanced prostate cancer (PCa) in many countries, but its effect on castration-resistant PCa (CRPC) is limited. We previously showed that resistance to bicalutamide results from activation of mechanistic target of rapamycin (mTOR). Interestingly, clinical trials testing combinations of the mTOR inhibitor RAD001 with bicalutamide were effective in bicalutamide-naïve CRPC patients, but not in bicalutamide-pretreated ones. Here we investigate causes for their difference in response. Evaluation of CRPC cell lines identified resistant vs sensitive in vitro models, and revealed that increased eIF4E(S209) phosphorylation is associated with resistance to the combination. We confirmed using a human-derived tumor xenograft mouse model that bicalutamide pre-treatment is associated with an increase in eIF4E(S209) phosphorylation. Thus, AR suppressed eukaryotic initiation factor 4E (eIF4E) phosphorylation, while the use of antiandrogens relieved this suppression, thereby triggering its increase. Additional investigation in human prostatectomy samples showed that increased eIF4E phosphorylation strongly correlated with the cell proliferation marker Ki67. Small interfering RNA-mediated knockdown (k/d) of eIF4E-sensitized CRPC cells to RAD001+bicalutamide, whereas eIF4E overexpression induced resistance. Inhibition of eIF4E phosphorylation by treatment with CGP57380 (an inhibitor of mitogen-activated protein kinase-interacting serine-threonine kinases MAP kinase-interacting kinase 1 (Mnk1/2), the eIF4E upstream kinase) or inhibitors of extracellular signal-regulated kinase 1/2 (ERK1/2), the upstream kinase-regulating Mnk1/2, also sensitized CRPC cells to RAD001+bicalutamide. Examination of downstream targets of eIF4E-mediated translation, including survivin, demonstrated that eIF4E(S209) phosphorylation increased cap-independent translation, whereas its inhibition restored cap-dependent translation, which could be inhibited by mTOR inhibitors. Thus, our results demonstrate that while combinations of AR and mTOR inhibitors were effective in suppressing tumor growth by inhibiting both AR-induced transcription and mTOR-induced cap-dependent translation, pre-treatment with AR antagonists including bicalutamide increased eIF4E phosphorylation that induced resistance to combinations of AR and mTOR inhibitors by inducing cap-independent translation. We conclude that this resistance can be overcome by inhibiting eIF4E phosphorylation with Mnk1/2 or ERK1/2 inhibitors.

Figure 1. Correlation between the responses of CRPC cells to bicalutamide (Casodex) and RAD001 and the expression and phosphorylation of various proteins

(A) MTT viability assay of PCa cell lines C4-2, 22Rv1 (Rv1), PC346C (not related to AR-null PC-3) and CWR-R1 (R1) treated with RAD001 (1nM) alone or in combination with 10 μM Bicalutamide (Caso) for up to 7 days. In C4-2 cells, bicalutamide and RAD001 individually reduced viability by 17% (p=0.065) and 61% (p-0.002) respectively, compared to control. However, the combination reduced viability by 69% (p=0.002), therefore improving the effects of both. Similarly, in 22Rv1 cells, neither bicalutamide (22%, p=0.26) nor RAD001 (38%, p=0.192) alone causes a significant viability reduction compared to control. However, the combination reduced viability by 68.7% (p=0.0259). Even in the resistant line CWR-R1, bicalutamide (12% decrease, p=0.004) improved the effects of RAD001 alone (36% decrease, p=0.0003) to 49.5% (p=0.0002); however, in PC-346C, bicalutamide made the effects of RAD001 (42.5%, p=0.023) worse (24.6%, p=0.004). Bicalutamide sensitive LNCaP cells were used as positive control. (Sensitivity to the drug combination was defined as >50% loss of viability). Results are shown in absorbance fold change to first day of treatment. (B) Western blots of C4-2, PC346C, CWR-R1, 22Rv1 and LNCaP cells without any treatment to show and compare basal levels of protein expression and phosphorylation between these cell lines. (C) MTT viability assay of C4-2 and PC346C cells challenged with the rapalogs BEZ235 (10nM) or INK128 (25nM) alone or in combination with Bicalutamide (Caso) for up to 7 days. Similar to RAD001, only C4-2, but not PC-346C, responded to the combination of BEZ235 and bicalutamide (89% reduction for C4-2, p=0.0008; 14.5% for PC-346C, p=0.123); however, both cell lines responded, at least partially, to the combination of INK128 and bicalutamide (94% reduction for C4-2, p=0.00013; 67.2% for PC-346C, p=0.006). (D) Western blots showing the mTOR pathway molecular profile of C4-2 and PC346C treated with RAD001, BEZ235 or INK128 for 48 hours.

Figure 2. Effect of bicalutamide and the mTORC1 inhibitor rapamycin on PSA levels and the phosphorylation of Akt and eIF4E in immunocompromised mice bearing CWR22 tumors

(A) Boxplot showing comparison of PSA scores of serum collected from mice in each arm of the study (Bicalutamide alone vs. Bicalutamide in combination with Rapamycin) from weeks 1 and 2 (n=6 per arm). (B) Left - Immunohistochemistry staining of CWR22 xenografts from nude mice treated with DMSO, Rapamycin, Bicalutamide or the combination (n=6 per arm). After end of treatment, mice were euthanized and the tumors excised. Formalin fixed paraffin-embedded tumor specimens were stained with anti-phospho-Akt (Ser473) antibody. Right – Boxplot of phospho-Akt in CWR22 xenografts from different treated mice demonstrating a slight but not significant increase in akt phosphorylation in rapamycin treated mice when compared to vehicle alone and a significant increase in phospho-akt in the dual treated mice when compared to control (p=0.025) and bicalutamide alone (p=0.022) (n=6 per arm). (C) Left – Immunohistochemistry staining of CWR22 xenografts from nude mice with the same treatments as above now blotted against phospho-eIf4E (S209). Right – Boxplot of phospho-eIF4E scores in CWR22 xenografts showing a sharp increase in phosphorylation of eIF4E in Bicalutamide treated mice. Investigators were not blinded to the outcomes in this experiment.

Figure 3. The AR is a negative regulator of eIF4E phosphorylation at S209

Western blots of C4-2 (A) and PC346C (B) cells treated with either bicalutamide alone or in combination with RAD001 and collected in different time points from 1 minute to 4 days. Different proteins of the mTOR pathway were blotted to show changes in expression and phosphorylation when treated with the drugs. It is noticeable an increase in P70S6K phosphorylation in both cell lines when treated with Bicalutamide alone, which goes in hand with previous findings from our laboratory where AR antagonists increase mTOR activity. The combination of an mTOR inhibitor with Bicalutamide ceases that activation as shown in both cells. We can also notice that phosphorylation of eIF4E remains the same overall in C4-2 but it’s increased in PC346C in both treatments. (C) Immunofluorescence of C4-2 cells treated for 48 hours with either vehicle (DMSO) or Bicalutamide and stained for phospho-eIF4E (FITC) or nuclear marker DAPI and both stains results overlapped (right) demonstrating increase in eIf4E phosphorylation in Bicalutamide treated cells. (D) Western blots of the normal prostate cell line PRSN1-1 and CRPC cells C4-2, C4-2b and PC346C demonstrating inverse correlation between AR expression and eIF4E phosphorylation. (E) MTT viability assay of PRNS1-1 (upper) or PRNS1-1 cells that were stable transfected with AR mutant T877A (lower) treated with different mTOR inhibitors up to 7 days showing no effect on viability of this cell line by any of the mTOR inhibitors unless AR was expressed. (F) Western blots of mTOR pathway proteins in PRNS1-1 cells transfected with empty vector, WT-AR or T877A mutant demonstrating decrease in phospho-eIF4E when AR is present in cells.

Figure 4. Correlation between eIF4E phosphorylation at S209 and Ki67 levels in PCa patient tissue obtained by prostatectomy

(A,B) left -Formalin fixed paraffin-embedded human localized PCa specimens obtained by prostatectomy were arranged in a tissue microarray and stained with either anti-eIF4E (A) or anti-phospho-eIf4E (B) antibody. All stains other than Ki67 were analyzed based on scores from 0–3, where 0 represents no staining and 3 represents 100% staining, whereas Ki67 staining was scored based on the actual number of nuclei within a specified area staining for the antigen. Right - Boxplots depicting the intensity of eIf4E or phospho-eIF4E in the different tissue samples demonstrate that the expression of eIf4E and phospho-eIF4E is more prominent in tumor samples when compared to benign tissue samples. (C) Left - IHC of tissue microarray from localized PCa specimens from two different patients stained with either anti-phospho-eIF4E or the proliferation marker anti-Ki67. Right – Boxplot depicting the intensity of phospho-eIF4E vs. the Median Ki67 demonstrating positive correlation between eIF4E phosphorylation at Serine 209 and cell proliferation.

Figure 5. eIF4E phosphorylation levels regulate proliferation in vitro

(A) Western blots of PC346C transfected with either control siRNA or eIF4E siRNA and treated with the different mTOR inhibitors alone or in combination with Bicalutamide (Caso) for 48 hours. (B and C) MTT viability assay of PC346C transfected with either control siRNA or eIF4E siRNA treated with RAD001, Bicalutamide or the combination. Neither RAD001 nor bicalutamide had significant effect on viability of PC-346C cells transfected with control siRNA (p=0.0755 for vehicle vs bicalutamide; p=0.0680 for vehicle vs RAD001) (Figure 5B); but in eIF4E knockdown cells RAD001 caused 62.1% loss in cell viability (p=0.0014), while in combination with bicalutamide, this effect increased to 80.8% (p=0.00098) (Figure 5C). (D) Western blots of C4-2 cells transfected with either empty vector or pHA-eIF4E and treated with the different mTOR inhibitors alone or in combination with Bicalutamide (Caso) for 48 hours. The eIF4E overexpressing cells demonstrate two bands representing eIF4E – an upper band which demonstrates the expression of the transfected plasmid (which is slightly larger than the endogenous eIF4E due to the HA tag) and a lower band representing the endogenous eIF4E. (E) MTT viability assay of C4-2 cells transfected with either an empty vector or pHA-eIF4E and treated with RAD001, collected up to day 6.

Figure 6. Activation of eIF4E phosphorylation at Ser209 by RAD001 and bicalutamide is mediated by an increase in ERK and p38MAPK activation

(A) C4-2 and PC-346C cells were treated for 48 hours with DMSO (vehicle), 10 μM bicalutamide, 1 nM RAD001 or both, then the cells were lysed and evaluated for the various proteins indicated. P-ERK indicates ERK(Y204/T202) and p-p38MAPK indicates p38MAPK(Y180/Y182). T-ERK and t-p38MAPK indicates total (phosphorylated and unphosphorylated) protein. (B) Luciferase assay of C4-2 cells transfected with plasmid expressing hPSA-luc and treated with vehicle, RAD001 (0.1, 1 or 10nM) or BEZ235 (5, 10 or 20nM). AR transcriptional activity was increased by all concentrations of RAD001 while it was decreased by all concentrations of BEZ235. (C) Western blots of C4-2 and PC346C treated with the control vehicle DMSO, the mTOR inhibitor RAD001 or the PI3K/mTOR dual inhibitor BEZ235 for 48 hours and blotted for proteins from the mTOR and MAPK pathways. (D) Western blots of 22Rv1 tumors extracted from athymic nu/nu mice subjected to sham-operation (intact) or castration. Results show an increase in phosphorylation of eIF4E, p38MAPK and ERK in castrated mice compared to intact. (E) Western blot of PC346C cells treated with either the mTOR inhibitor RAD001, ERK1/2 inhibitor PD98059 or the p38MAPK inhibitor SB203580 for 48 hours. (F) MTT viability assay of PC346C cells treated with RAD001, PD98059, SB203580 and the combinations. The combination of ERK and P38MAPK inhibitor sensitized PC346C cells to the mTOR inhibitor RAD001. Results are shown in absorbance fold change to first day of treatment.

Figure 7. Suppression of eIF4E phosphorylation at Ser209 by a Mnk1/2 inhibitor sensitizes CRPC cells to RAD001

(A) Western blots of PC346C cells treated with the Mnk1/2 inhibitor CGP57380 demonstrating decrease in eIF4E phosphorylation over 4 days. The effect of CGP57380 (5μM) in PC-346C cells is biphasic and caused an initial decrease at 2 hours followed by an increase at 24 hours (less than initial) which was followed by a subsequent decrease after 72 hours. (B) Western blots of C4-2 cells treated for 48 hours with CGP57380, RAD001 or the combination and blotted against phospho and total eIf4E, total Mnk and the loading control tubulin. (C) Immunofluorescence of C4-2 and PC346C cells treated for 48 hours with the Mnk inhibitor CGP57380, the mTOR inhibitor RAD001 and the combination and stained for phospho-eIF4E demonstrating the increase in phosphorylation of eIF4E when treated with RAD001 and the decrease of the same effect in CGP57380 treated cells. The combination treatment also shows inhibition of the increase of phospho-eIF4E induced by RAD001 if CGP57380 is present. (D) MTT viability assay of C4-2, PC346C, 22Rv1 and CWR-R1 cells treated with CGP57380, RAD001 or the combination compared to control for up to 7 days. All cell lines had viability decreased in presence of the Mnk inhibitor CGP57380 (Less effective in R1 cells) and this effect was augmented with the combination. Results are shown in absorbance fold change to first day of treatment and sensitivity to the drug combination was defined as >50% loss of viability. (E) PI single stain flow cytometry of PC346C cells treated with RAD001, CGP57380 or the combination for analysis of cell cycle showing increase in G1 phase arrest in these cells with both drugs separately and even more with the combination. (F) Western blots of different mTOR pathway proteins of C4-2 and PC346C treated for 48 hours with RAD001, CGP57380 or the combination.

Figure 8. Bicalutamide-induced eIF4E phosphorylation at Ser209 switches translation from a cap-dependent to a cap-independent mechanism resistant to RAD001, whereas the Mnk inhibitor CGP57380 reverses this switch

(A) Western blots of C4-2 and PC346C treated with DMSO, RAD001, Bicalutamide and the combination for 48 hours and blotted for eIF4E targets. In C4-2 the treatment with RAD001 or RAD+Bicalutamide had an effect on survivin expression but not in PC346C cells. (B) Western blots of C4-2 and CWR-R1 transfected with either control siRNA or eIF4E siRNA for 48 hours. EIF4E knock down affects surviving expression in both cell lines. (C) Western blots of C4-2 and PC346C treated with either vehicle or the Mnk inhibitor CGP57380 for 48 hours. Again we see eIF4E phosphorylation decrease corresponding with decrease in survivin expression in C4-2 but not in PC346C. (D-E) C4-2 (D) and PC346C (E) were transfected with the pFL-EMCV-IRES-RL construct and treated with RAD001, Bicalutamide, CGP57380 and the possible combinations for 48 hours. Readings represent a mean±S.D. of the ratio of Firefly/Renilla from triplicates.