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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.

To identify biomarkers that discriminate the aggressive forms of prostate cancer, we performed gene expression profiling of prostate tumors using a genetically engineered mouse model that recapitulates the stages of human prostate cancer, namely Nkx3.1; Pten mutant mice. We observed a significant deregulation of the epidermal growth factor and mitogen-activated protein kinase (MAPK) signaling pathways, as well as their major downstream effectors--the activator protein-1 transcription factors c-Fos and c-Jun. Forced expression of c-Fos and c-Jun in prostate cancer cells promotes tumorigenicity and results in activation of extracellular signal-regulated kinase (Erk) MAPK signaling. In human prostate cancer, up-regulation of c-Fos and c-Jun proteins occurs in advanced disease and is correlated with Erk MAPK pathway activation, whereas high levels of c-Jun expression are associated with disease recurrence. Our analyses reveal a hitherto unappreciated role for AP-1 transcription factors in prostate cancer progression and identify c-Jun as a marker of high-risk prostate cancer. This study provides a striking example of how accurate mouse models can provide insights on molecular processes involved in progression and recurrence of human cancer.

Prostate cancer is the second leading cause of cancer-related deaths in men. Activation of MAP kinase signaling pathway has been implicated in advanced and androgen-independent prostate cancers, although formal genetic proof has been lacking. In the course of modeling malignant melanoma in a tyrosinase promoter transgenic system, we developed a genetically-engineered mouse (GEM) model of invasive prostate cancers, whereby an activating mutation of BRAF(V600E)--a mutation found in approximately 10% of human prostate tumors--was targeted to the epithelial compartment of the prostate gland on the background of Ink4a/Arf deficiency. These GEM mice developed prostate gland hyperplasia with progression to rapidly growing invasive adenocarcinoma without evidence of AKT activation, providing genetic proof that activation of MAP kinase signaling is sufficient to drive prostate tumorigenesis. Importantly, genetic extinction of BRAF(V600E) in established prostate tumors did not lead to tumor regression, indicating that while sufficient to initiate development of invasive prostate adenocarcinoma, BRAF(V600E) is not required for its maintenance.

In this report, we have investigated the relationship between androgen levels and prostate tumorigenesis in Nkx3.1; Pten mutant mice, a genetically engineered mouse model of human prostate cancer. By experimentally manipulating serum levels of testosterone in these mice for an extended period (i.e., 7 months), we have found that prolonged exposure of Nkx3.1; Pten mutant mice to androgen levels that are 10-fold lower than normal (the "Low-T" group) resulted in a marked acceleration of prostate tumorigenesis compared with those exposed to androgen levels within the reference range (the "Normal-T" group). We found that prostate tumors from the Low-T mutant mice share a similar gene expression profile as androgen-independent prostate tumors from these mutant mice, which includes the deregulated expression of several genes that are up-regulated in human hormone-refractory prostate cancer, such as Vav3 and Runx1. We propose that exposure to reduced androgens may promote prostate tumorigenesis by selecting for molecular events that promote more aggressive, hormone-refractory tumors.

Purpose: Epidemiologic studies have shown that reduced levels of vitamin D represent a major risk factor for prostate cancer. In this report, we have examined the efficacy of 1alpha,25-dihydroxyvitamin D(3) (1,25 D(3)) as a chemopreventive agent using Nkx3.1; Pten mutant mice, which recapitulate stages of prostate carcinogenesis from prostate intraepithelial neoplasia (PIN) to adenocarcinoma., Experimental Design: 1,25 D(3) (or vehicle) was delivered continuously to Nkx3.1; Pten mutant or control mice for a 4-month period beginning before (precancerous cohort) or after (cancerous cohort) these mice developed PIN. At the conclusion of the study, the mice were analyzed for the occurrence of PIN and/or cancer phenotypes by histologic analyses and immunostaining using known markers of cancer progression in these mice., Results: We found that sustained delivery of 1,25 D(3) to the Nkx3.1; Pten mutant mice resulted in a significant reduction in the formation of PIN while having no apparent effect on the control mice. Furthermore, 1,25 D(3) was maximally effective when delivered before, rather than subsequent to, the initial occurrence of PIN. We further show that this 1,25 D(3)-mediated inhibition of PIN was coincident with up-regulation of vitamin D receptor expression in the prostatic epithelium of the mutant mice, as well as in CASP prostate epithelial cell lines developed from these mice, while having no effect on androgen receptor expression or androgen receptor signaling., Conclusion: Our findings show the value of chemoprevention studies using Nkx3.1; Pten mutant mice, particularly for evaluating the efficacy and underlying mechanisms of potential agents and to gain insights about the optimal timing of their delivery. In particular, our study predicts that vitamin D may have differential effects during early-stage versus late-stage disease and that it is more likely to be beneficial if delivered either before the overt manifestation of clinically detectable disease or during the earliest disease stages, rather than in advanced disease. Thus, our findings support the assessment of vitamin D analogues for chemoprevention in clinical trials targeting patients with early-stage disease and also establish molecular markers that can be used in such trials to determine biological activity and to optimize further clinical trials.

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.

Although androgen deprivation therapy is a widely used treatment for patients with advanced prostate cancer, it ultimately results in the emergence of a hormone-refractory disease that is invariably fatal. To provide insights into the genesis of this disease, we have employed an in vivo model to investigate how and when prostate epithelial cells can acquire the ability to survive and proliferate in the absence of androgens. In particular, we have been studying the evolution of androgen independence in Nkx3.1; Pten mutant mice, which develop prostatic intraepithelial neoplasia and adenocarcinoma as a consequence of aging, as well as androgen-independent phenotypes following castration. We now find that the prostate epithelial cells from these Nkx3.1; Pten mutant mice are capable of surviving and proliferating in the absence of androgens and that they develop androgen-independent phenotypes well before they display overt prostatic intraepithelial neoplasia or cancer phenotypes. Our findings in this mouse model show that acquisition of androgen independence can be uncoupled from overt cancer progression and raise the possibility that hormone-refractory disease can arise at early stages of prostate carcinogenesis.

Despite the significance of oxidative damage for carcinogenesis, the molecular mechanisms that lead to increased susceptibility of tissues to oxidative stress are not well-understood. We now report a link between loss of protection against oxidative damage and loss-of-function of Nkx3.1, a homeobox gene that is known to be required for prostatic epithelial differentiation and suppression of prostate cancer. Using gene expression profiling, we find that Nkx3.1 mutant mice display deregulated expression of several antioxidant and prooxidant enzymes, including glutathione peroxidase 2 and 3 (GPx2 and GPx3), peroxiredoxin 6 (Prdx6), and sulfyhydryl oxidase Q6 (Qscn6). Moreover, the formation of prostatic intraepithelial neoplasia in these mutant mice is associated with increased oxidative damage of DNA, as evident by increased levels of 8-hydroxy-2'-deoxyguanosine. We further show that progression to prostate adenocarcinoma, as occurs in compound mutant mice lacking Nkx3.1 as well as the Pten tumor suppressor, is correlated with a further deregulation of antioxidants, including superoxide dismutase enzymes, and more profound accumulations of oxidative damage to DNA and protein, the latter manifested by increased levels of 4-hydroxynonenal. We propose that the essential role of Nkx3.1 in maintaining the terminally differentiated state of the prostate epithelium provides protection against oxidative damage and, thereby, suppression of prostate cancer. Thus, our findings provide a molecular link between a gene whose inactivation is known to be involved in prostate carcinogenesis, namely Nkx3.1, and oxidative damage of the prostatic epithelium.

In human prostate cancer, the frequent down-regulation of p27(kip1) protein expression is correlated with poor clinical outcome, yet p27(kip1) rarely undergoes mutational inactivation. Here, we investigate the consequences of reducing or eliminating p27(kip1) function for prostate carcinogenesis in the context of a mouse modeling lacking the Nkx3.1 homeobox gene and the Pten tumor suppressor. Unexpectedly, we find that triple mutant mice heterozygous for a p27(kip1) null allele (Nkx3.1(+/- or -/-); Pten(+/-); p27(+/-)) display enhanced prostate carcinogenesis, whereas mice that are homozygous null for p27(kip1) (Nkx3.1(+/- or -/-); Pten(+/-); p27(-/-)) show inhibition of cancer progression. Expression profiling reveals that Cyclin D1 is highly up-regulated in compound p27(kip1) heterozygotes, but is down-regulated in the compound p27(kip1) homozygous mutants. Using RNA interference in prostate cancer cell lines with distinct p27(kip1) gene doses, we show that prostate tumorigenicity depends on levels of p27(kip1) and that the consequences of p27(kip1) gene dosage can be attributed, in part, to altered levels of Cyclin D1. Our findings suggest that p27(kip1) possesses dosage-sensitive positive as well as negative modulatory roles in prostate cancer progression.