Combinatorial activities of Akt and B-Raf/Erk signaling in a mouse model of androgen-independent prostate cancer

Abstract

Androgen independence is responsible for most prostate cancer lethality, yet currently there are no effective clinical treatments. We have been investigating the mechanisms underlying androgen-independent prostate cancer in Nkx3.1;Pten mutant mice, which display salient features of the disease, including a requirement for wild-type androgen receptor (AR) signaling. We now demonstrate that the Akt and Erk MAP kinase signaling pathways are activated in androgen-independent lesions of these mice. Forced activation of either Akt or Erk signaling in an androgen-responsive prostate cancer cell line promotes hormone-independent but AR-dependent growth in culture. Although these pathways act additively in culture, they act synergistically in vivo to promote tumorigenicity and androgen independence in the context of the prostate microenvironment. We propose that androgen independence emerges by means of epithelial-stromal competition, in which activation of Akt and Erk promotes AR activity in the prostate epithelium while counteracting antagonistic effects of the stroma.

Fig. 1.

Activation of Akt and Erk in androgen-independent lesions of Nkx3.1;Pten mutant mice. Shown are sections from the anterior prostate of wild-type (Nkx3.1^+/+;Pten^+/+) or mutant (Nkx3.1^+/−;Pten^+/−) mice castrated at 10 months and analyzed at 15 months. Sections were stained with H&E or analyzed by immunohistochemistry for AR or activated forms of Akt (p-Akt) or Erk (p-Erk). (Scale bars: 100 μm.)

Fig. 2.

Androgen independence in Nkx3.1;Pten requires AR signaling. Tissue recombinants were generated with adult prostate epithelium from wild-type (Nkx3.1^+/+;Pten^+/+) or mutant (Nkx3.1^+/−;Pten^+/−) mice and mesenchyme from rat embryonic urogenital sinus and grown in nude male hosts that were castrated or mock-castrated and/or implanted with a flutamide pellet (100 mg). (A–H) Representative images indicating the tissue recombinant (recomb) and host kidney. (I–X) Tissue sections showing H&E staining or immunohistochemistry with the indicated antibodies.

Fig. 3.

Activation of Akt and B-Raf/Erk signaling pathways is additive for androgen independence in cell culture. Retroviral vectors expressing *Akt or *B-Raf or a control (vector) were introduced individually or in combination into the androgen-responsive cell line CASP 2.1, and cells were grown in conditions of androgen deprivation. (A) Western blot analyses of CASP 2.1 protein extracts using the indicated antibodies. (B and C) Proliferation (B) and anchorage independence (C) assays after growth in media depleted of androgens or with androgens (+ DHT). Shown are representative assays done in triplicate; error bars represent one standard error. (D) Western blot analyses of cells expressing *Akt plus *B-Raf and treated with 50 mM rapamycin and/or 50 mM U0126 (as indicated). (E–G) Cell-cycle analyses of CASP 2.1 cells expressing *Akt and/or *B-Raf. Shown are relative percentages of cells in G₂/M phase compared with the vector or vehicle control. (Insets) FACS data and percentage of cells in G₂/M for representative samples (indicated by a and b). Cells were grown in androgen-depleted media without additions (E) or with 20 mM flutamide (F) or 50 nM rapamycin and/or 50 mM U0126 (G) as indicated. Experiments were performed a minimum of three times in triplicate; the standard error is shown. ∗∗, P < 0.01 (between a and b in each set).

Fig. 4.

Activation of Akt and B-Raf/Erk signaling pathways is synergistic for androgen independence in vivo. (A–F) Orthotopic tumor assays. CASP 2.1 cells expressing *Akt and/or *B-Raf (experimental) or a control vector (vector) were grown orthotopically in the dorsal prostates of nude male mice that were castrated or mock-castrated. Shown are representative images of dorsal prostate lobes showing the experimental or vector-injected sides. Note that the prostates infected with *Akt and *B-Raf completely overgrew the urogenital system and thus the vector control is not evident. (G–N) Analyses of apoptosis in culture and in vivo. CASP 2.1 cells expressing the vector or *Akt plus *B-Raf were grown in culture (G and H) or orthotopically in the prostate (K–N). TUNEL assays were performed 2 days after androgen deprivation of the cells in culture or 2 days after castration of the host.

Fig. 5.

Epithelial–stromal competition model of androgen independence. Emergence of androgen independence is coincident with activation of Akt (red) or Erk MAP kinase (blue) or both (purple) in prostate epithelial cells. During cancer progression (arrow), these “activated” prostate epithelial cells promote the growth of the tumor cells under conditions of androgen deprivation while counteracting the negative influences of the prostate stroma.