Targeting AKT/mTOR and ERK MAPK signaling inhibits hormone-refractory prostate cancer in a preclinical mouse model

Abstract

The AKT/mammalian target of rapamycin (AKT/mTOR) and ERK MAPK signaling pathways have been shown to cooperate in prostate cancer progression and the transition to androgen-independent disease. We have now tested the effects of combinatorial inhibition of these pathways on prostate tumorigenicity by performing preclinical studies using a genetically engineered mouse model of prostate cancer. We report here that combination therapy using rapamycin, an inhibitor of mTOR, and PD0325901, an inhibitor of MAPK kinase 1 (MEK; the kinase directly upstream of ERK), inhibited cell growth in cultured prostate cancer cell lines and tumor growth particularly for androgen-independent prostate tumors in the mouse model. We further showed that such inhibition leads to inhibition of proliferation and upregulated expression of the apoptotic regulator Bcl-2-interacting mediator of cell death (Bim). Furthermore, analyses of human prostate cancer tissue microarrays demonstrated that AKT/mTOR and ERK MAPK signaling pathways are often coordinately deregulated during prostate cancer progression in humans. We therefore propose that combination therapy targeting AKT/mTOR and ERK MAPK signaling pathways may be an effective treatment for patients with advanced prostate cancer, in particular those with hormone-refractory disease.

Figure 1. Inhibition of AKT/mTOR and ERK MAPK signaling pathways with rapamycin and PD0325901.

(A) Diagram of the experimental strategy. Nkx3.1; Pten mutant mice develop low-grade and high-grade PIN (LGPIN and HGPIN, respectively) and ultimately adenocarcinoma as a consequence of aging, as well as androgen independence following castration. The trial design entailed enrolling androgen-intact or androgen-ablated mutant (or control) mice at approximately 10 months of age randomly into groups that were treated with rapamycin and/or PD0325901 (or vehicle) for 21 days (5 days on/2 days off), after which the mice were sacrificed (Sac) for analyses of end points (i.e., histology, prostate weights, cellular proliferation, immunohistochemistry, and Western blot analyses; Figures 3–6 and Table 1). AD, androgen-dependent; AI, androgen-independent. (B) Rapamycin and PD0325901 inhibit their respective targets in the prostate for up to 24 hours. Western blot analyses were performed using protein extracts prepared from the dorsolateral prostate of Nkx3.1^+/—Pten^+/— mutant mice (10 months) treated with rapamycin plus PD0325901 for the times indicated. Each group had 3 mice; Western blot analyses were done with at least 2 independent mice in each group, and representative samples are shown. (C–N) Rapamycin and PD0325901 lead to inhibition of target proteins in mouse prostate tissues in vivo. Immunohistochemical analyses were performed using the indicated antibodies on sections from the anterior prostate of Nkx3.1^+/—Pten^+/— mutant mice (10 months; androgen-intact) treated with rapamycin and/or PD0325901 (or vehicle) as indicated for 1 week. Scale bar: 100 μm.

Figure 2. Rapamycin and PD0325901 display strong synergism in cell culture.

(A–D) Analyses of IC₅₀ plots for the single agents (A and B) and the combination agents (C and D). (E) Graphic representation of the CI for rapamycin and PD0325901. These data are shown for CASP 1.1 cells; similar results were obtained for CASP 2.1 cells (not shown). Note that CI values were well below 0.1, indicating strong synergism of the drugs. (F and G) Bim is upregulated in response to drug treatment. CASP 1.1 or CASP 2.1 cells were transfected with a control or Bim siRNAi, followed by treatment with vehicle or the indicated compounds in the medium for 48 hours. (F) Western blot analyses done on whole cell extracts using the indicated antibodies or a control for protein loading (Ponceau S staining). (G) Results of MTT assays, indicating the enhanced cell survival in the drug-treated cells following treatment with Bim siRNAi. The P values compare the control (siCont) and the Bim siRNAi (siBim) in each group. Data are expressed as mean ± SEM.

Figure 3. Modest efficacy of rapamycin and PD0325901 for treatment of androgen-dependent prostate cancer.

(A) Diagram of the experimental strategy, as per Figure 1. (B–U) Representative tissue sections from the Nkx3.1^+/—Pten^+/— mutant mice treated with the single agent or combination of agents, showing histological phenotype (H&E) and immunostaining for the antibodies indicated. H&E analyses were performed on all experimental mice in each group; immunohistochemistry was done on a minimum of 4 animals from each group; representative data are shown. Scale bars: 100 μm.

Figure 4. Profound efficacy for prostate histology following treatment of androgen-independent prostate cancer with rapamycin and PD0325901.

(A) Diagram of the experimental strategy, as per Figure 1. (B–U) Representative tissue sections from the androgen-ablated Nkx3.1^+/—Pten^+/— mutant mice treated with the single or combination of agents, showing histological phenotype (H&E) and immunostaining for the antibodies indicated. In these and all subsequent experiments, H&E analysis was performed on all experimental mice in each group; immunohistochemistry was done on a minimum of 4 animals from each group; representative data are shown. Scale bars: 100 μm.

Figure 5. Combination therapy is antitumorigenic for treatment of androgen-independent prostate cancer in Nkx3.1; Pten mutant mice.

Comparison of the consequences of single agent versus combination therapy for treatment of androgen-dependent (A–D) or androgen-independent (E–H) mutant mice. (A and E) Diagram of the experimental strategy, as per Figure 1. (B and F) Prostate tissue weights, showing mean ± SEM, with P value indicated. (C and G) Percentage of proliferating cells as determined by Ki67 staining, showing mean ± SEM, with P value indicated. (D and H) Western blots of protein extracts from prostate tissues following treatment. Western blotting was done using a minimum of 3 independent mice in each group; representative data are shown; total ERK is shown as a control for protein loading.

Figure 6. Combination therapy is antitumorigenic in tissue recombination models from the Nkx3.1; Pten mutant mice.

(A and I) Diagram of the experimental strategy. Tissue recombinants were made using prostate epithelium from Nkx3.1; Pten mutant or wild-type mice and rat embryonic mesenchyme and grown in nude mice for 1 month. Following 1 month of growth, the nude mice were either left intact (i.e., to model treatment of androgen-dependent prostate cancer) or castrated (i.e., to model treatment of androgen-independent prostate cancer). Mice received rapamycin and/or PD0325901 (or vehicle) for 21 days (5 days on/2 days off), following which the mice were sacrificed for analyses of end points (i.e., histology, prostate weights, and immunohistochemistry). (B–G and J–O) Representative tissue sections from the tissue recombinants made from the Nkx3.1^+/—Pten^+/— prostate epithelium showing histological phenotype (H&E) and immunostaining for p-S6 and p-ERK, as indicated. Scale bars: 100 μm. (H and P) Weights of the tissue graphs, showing mean ± SEM, with P value indicated. Recomb, recombinant.

Figure 7. Combination therapy is antitumorigenic in adjuvant therapy of hormone-refractory prostate cancer.

(A–I) Studies in the whole animal. (A) Diagram of the experimental strategy. Strategy is similar to that in Figure 1; however, unlike the androgen-independent treatment group, Nkx3.1^+/—Pten^+/— mutant mice were castrated immediately prior to receiving the rapamycin and/or PD0325901 (or vehicle) treatment. (B–G) Representative tissue sections from the Nkx3.1^+/—Pten^+/— mutant mice showing histological phenotype (H&E) and immunostaining for p-S6 and p-ERK, as indicated. (H) Prostate tissue weights, showing mean ± SEM, with P value indicated. (I) Percentage of proliferating cells as determined by Ki67 staining, showing mean ± SEM, with P value indicated. (J–Q) Studies done in a complementary tissue recombination model. (J) Diagram of the experimental strategy. (K–P) Representative tissue sections from the tissue recombinants made from the Nkx3.1^+/—Pten^+/— prostate epithelium showing histological phenotype (H&E) and immunostaining for p-S6K and p-ERK, as indicated. (Q) Weights of the tissue graphs, showing mean ± SEM, with P value indicated. Scale bars: 100 μm.

Figure 8. AKT/mTOR pathway activation is associated with human prostate cancer progression and correlated with activation of ERK MAPK.

(A–L) Expression of PTEN and components on the AKT/mTOR signaling pathway in human normal and primary prostate cancer samples. Representative adjacent sections from the specimens used for the TMAs show staining for the indicated proteins. Shown are examples of benign prostatic tissue (Normal), a tumor without activation of the PTEN/AKT/mTOR pathway (Tumor 1), and a tumor with activation of this pathway (Tumor 2). Note that the expression of PTEN is inversely correlated with expression of p-AKT, p-mTOR, and p-S6. (M–O) Expression of p-ERK activation on semiadjacent sections of the same specimens. Note that, in these samples, p-ERK activation is well correlated with activation of components of the AKT/mTOR signaling pathway. Scale bar: 100 μm.