Do phytonutrients affect prostate tumor progression?

Abstract
Key Personnel
Specific Objectives

Abstract
Great interest exists among consumers to use dietary supplements that will reduce their risk of cancer. Several plant-derived compounds have been suggested to prevent, and to potentially be useful in treating prostate cancer; however, the efficacy and safety of these compounds has not been rigorously tested. This is due in part to the lack of both adequate animal models for prostate cancer and a systematic approach to the identification of cellular mechanisms through which botanicals affect prostate tumor progression. The objective of this propject is to characterize the responses of key prostate tumor biomarkers to phytonutrients and to provide mechanisms for these responses. These biomarkers are the genetic and epigenetic changes, as well as the signal transduction pathways, which influence cell division and gene expression in the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) tumor mouse model. We will study phytonutrients, which are structurally related to phytoestrogens, and ask if they act through estrogen receptor-dependent pathways by using estrogen receptor (ER)-deficient mice (ERαKO, ERβKO and ERαKO/ERβKO) expressing the TRAMP transgene. We will test the effects of specific phytoestrogens in these three unique transgenic animals. We will characterize: 1) prostate tumor progression, as assessed by quantity, distribution, histological appearance, and the nature of metastases; 2) expression of androgen receptor (AR), ERα, and ERβ; 3) differential methylation of prostate tumor-related genes; 4) duplication and deletion of genetic loci known to be important in the progression of human prostate cancer; 5) expression of signaling-pathways for mitogen-activated protein kinase (MAPK), for insulin-like growth factor-I (IGF-I), and for protein kinase C (PKC). In previous work we have developed ERα-KO/TRAMP mice and documented the occurrence and progression of prostate tumors relative to ER-WT/TRAMP mice. Exciting preliminary data show that the onset of prostate tumors is earlier, but that progression is slowed in ERαKO/TRAMP mice. This suggests that ERα-dependent mechanisms influence specific steps in tumor progression. Unraveling these steps and characterizing how they are altered by dietary compounds, such as phytoestrogens, is the primary aim of the project. These studies will provide fundamental insights into prostate tumor biology and assess the roles of botanical dietary supplements in modifying the incidence and progression of prostate tumors. The information gained from these studies will contribute to the design of clinical trials using botanicals to alter the course of human prostate cancer. Potential clinical trials are proposed as Pilot Projects.

Key Personel
Dennis Lubahn — Principal Investigator
Kevin Fritsche — Co-principal Investigator
Cindy Besch-Williford — Co-principal Investigator
William Folk — Co-principal Investigator

Specific Objectives
The overall objective of this project is to characterize the response of key prostate tumor biomarkers to phytonutrients in the spontaneous prostate tumor mouse model (TRAMP). The primary phytonutrients to be examined will be potential phytoestrogens. We will determine if these phytonutrients are mediating estrogen receptor-dependent responses important in prostate cancer by using the estrogen receptor-alpha (ERα) and -beta (ERβ)-deficient or knockout mouse models (ERαKO, ERβKO and ERαKO/ERβKO) that are expressing the TRAMP transgene. Our hypothesis is that phytonutrients, particularly those with estrogenic activity, affect prostate tumor progression via both estrogen receptor-dependent and independent mechanisms. We propose to define phytonutrient effects on specific cellular mechanisms, which are believed to be important in prostate cancer. We will, thus, establish a system that will allow the effects on cancer of botanical ingredients to be assessed, in particular for prostate cancer. The collaboration among several Co-PIs promotes an interdisciplinary approach to study several prostate tumor markers. The experimental design with three animal models provided by the Animal/Nutrition Core will be to expose animals to phytonutrients in utero and throughout life in order to mimic human exposure to a dietary nutrient that is consumed as part of the normal diet or as a daily supplement. Female mice will be fed diets containing one phytonutrient for at least one week prior to breeding and throughout pregnancy and lactation. Offspring from the females will nurse their respective mothers until weaning; those pups of the desired genotype will then be fed the maternal diet for the duration of the experiment.

The transgenic animals generated by the Animal / Nutrition Core will allow identification of estrogen receptor a and/or b-dependent and -independent mechanisms for the dietary phytonutrients. The animal models to be used for Aims 1-3 are:
1. ERαKO; ERαKO/TRAMP, ERαWT, ERαWT/TRAMP
2. ERβKO, ERβKO/TRAMP, ERβWT, ERβWT/TRAMP
3. ERαKO+ERβKO, ERαKO+ERβKO/TRAMP, ERαWT+ERβWT, ERαWT+ERβWT/TRAMP

Objective 1
To determine the role of ERα in mediating effects of dietary phytonutrients in prostate tumor development. Specific prostate biomarkers will be measured at intervals during tumor progression in ERαKO and wild type mice (with or without the TRAMP transgene) fed plant phytoestrogenic compounds. The biomarkers to be measured include: prostate tumor progression, quantity, distribution and histological appearance, and characterization of metastases (Besch-Williford); quantitation of androgen receptor, ERα, and ERβ (Lubahn); differential methylation of prostate tumor-related genes (Lubahn and Huang); heterogeneity and chronology of the duplication and deletion of specific genetic loci known to be important in prostate cancer (Folk and Huang), and alteration of the urokinase plasminogen activator (uPA) and mitogen-activated protein kinase (MAPK) signaling pathways (Folk); alteration of the insulin-like growth factor-I (IGF-I) and protein kinase C (PKC) signaling pathways (MacDonald).

Objective 2
To determine the role of ERβ in mediating effects of dietary phytonutrients in prostate tumor development. Specific prostate biomarkers described in Objective 1 will be measured in ERβKO and wild type mice (with or without the TRAMP transgene) fed phytoestrogenic compounds.

Objective 3
To determine the function of the primary estrogen receptors in mediating effects of dietary phytonutrients in prostate tumor development. Specific prostate biomarkers described in Objective 1 will be measured in double knockout ERαKO/ERβKO or wild type mice (with or without the TRAMP transgene) fed phytoestrogenic compounds.