The NCCAM Intramural Endocrine laboratory is focused on the effects of selected androgenic (DHEA; dehydroepiandrosterone), estrogenic (phytoestrogens) or other dietary supplements and/or natural products on neoplastic prostate epithelial and stromal cell growth, gene expression, including signaling between stromal and epithelial cells and the intracellular signal transduction pathways. As a precursor to both estrogen and testosterone, DHEA excess may pose a potential cancer risk in hormone-responsive tissues such as the prostate. We have investigated effects of DHEA on epithelial and stromal cells from normal and cancer tissues, grown separately and in co-culture. Prostate cancer epithelial cells exhibit differential responses, to DHEA, depending in part upon their androgen receptor (AR) status. Prostate cancer cells with a mutated AR and are responsive to DHEA treatment as measured by cell proliferation, and expression of prostate specific antigen (PSA), and IGF axis proteins. In contrast, prostate cancer cells, with a normal AR, are minimally responsive. Prostatic primary stromal cells (6S cells) were responsive to androgens, such as dihydrotestosterone (DHT) by increasing secretory IGF-1, whereas DHEA did not produce the same effect. ? ? Important mechanisms of DHEA effects on prostate cells were revealed when cancer cells (with the normal AR) were combined in culture with the stromal cells. When cancer cells were grown in the presence of stromal cells, DHEA stimulated epithelial PSA protein levels to that achieved by DHT-induction. DHEA-treated stromal cells were able to metabolize DHEA to androgenic ligands thereby inducing increased epithelial PSA production in these cocultures. The addition of TGFbeta -1 to the cocultures increased PSA protein secretion 2-4 times over DHEA alone and increased gene expression up to 50-fold. Also in cocultures of LAPC-4 + 6S cells, TGFbeta -1 increased metabolism of DHEA to testosterone. Addition of TGFbeta models a reactive prostate stroma as is associated with the cancer tissue microenvironment. These results indicate that in cancer tissues compared to normal prostate, there is promotion of metabolism of DHEA to androgenic ligands, resulting in increased growth and PSA production. Red Clover contains various isoflavones, including genistein, daidzein biochanin A, a formononetin. Red clover isoflavones decreased PSA protein and gene expression and T metabolism induced by TGFβ +DHEA in prostate LAPC-4/6S cocultures in a dose dependent manner. In this coculture model of endocrine-immune-paracrine interactions in the prostate, provides a tool for discovery of natural products or traditional medicines that may be involved in stromalepithelial cell interactions especially altering paracrine hormonal signals.? ? Stromal targets are being identified by gene expression microarray analysis (Affymetrix U133 chips) of DHEA vs androgen (R1881)-treated stromal cells in coculture vs. monoculture with cancer epithelial cells and signaling mechanisms elaborated. Also identification of stromal paracrine mediators was probed using SuperArray Real Time RT-PCR technology. Among other genes found to be upregulated, there were IGF-1, FGF-1 and FGF-7. These and other selected growth factors will be further explored as targets for stromal regulation of epithelial cell function.? ? Hydroxysteroid dehydrogenase (HSD) metabolizing enzymes were targeted by siRNA knockdown in the stromal cells of the cocultures to determine effects of TGFbeta on metabolism of DHEA. Western blot for 3b-HSD, 17b-HSD type 1, and 17b-HSD type 5 showed significant knockdown of these metabolizing enzymes in 6S cells treated with siRNA against the respective enzymes compared with 6S cells treated with control siRNA. There was an accompanying significant decrease in PSA levels in cocultured epithelial cells. Thus 3b-HSD, 17b-HSD(1) and 17b-HSD(5) all play significant roles in DHEA metabolism in reactive stroma by the read-out of the production of PSA levels in LAPC4cells.? ? A parallel study explored role of the natural product, lycopene, in modulating DHT-induced intracellular and extracellular stromal signaling and the resulting effects on epithelial cell signal transduction and cell death. Comparative studies of paracrine signaling from reactive versus normal stroma were carried out using normal human prostate epithelial (NPE) cells. In this study, lycopene, in dietary concentrations, reversed DHT effects of 6S cells on induced NPE cell death; decreased 6S cell IGF-I production by reducing AR and beta-catenin nuclear localization; and inhibited IGF-I-stimulated NPE and PREC growth by attenuating IGF-I's effects on serine phosphorylation of Akt and GSK3 beta and tyrosine phosphorylation of GSK3. This study provides possible mechanisms of lycopene prevention of prostate cancer. ? ? DHEA metabolism to estrogens can play an important role in prostate functional ERbeta is an important target in prostate cells for endogenous and exogenous estrogens and plays a role in modulating androgen activity. Steroid hormones cross-talk with Wnt/beta- catenin pathway and may interact with ER beta in prostate cells. The signal transduction pathways involved in DHEA and its estrogenic metabolites were investigated in prostate cancer cells that are AR(-) but ER beta positive. The possible pathway is that DHEA is metabolized to 3beta-Adiol or E2, which binds to ERbeta. This binding changes ERbeta associations. Direct associations of ERbeta with Galpha were suggested in this study and proved in other studies. Changing associations of Galpha may contribute to beta-catenin stabilization and increase of the signaling. The study reveals DHEA or its metabolites activate beta-catenin/TCF signaling through ERbeta/Galpha interactions and gives a new insight of the non-genomic effect of these steroid hormones.? ? We have been elaborating the downstream signaling of Wnt and the interactions between adipose differentiation-related protein (ADRP), Dishevelled (Dvl) and Axin complexes and subsequent rapid inactivation of GSK3 beta/Axin complexes. Thus, rapid delivery of Wnt-signaling from Frizzled (Fz) to Axin complexes is mediated by protein association changes, resulting in inactivation of GSK3 beat/Axin complexes and stabilization of beta-catenin Important also in the signaling is the role of the two G-alpha subunits, G-alpha-o and G-alpha-q, delivering Wnt-signaling from Fz to Dvl. These signaling pathways further elaborate intracellular response to steroid hormones in prostate cells. ? ? We hypothesize that DHEA exerts minimal effects in the normal prostate, whereas in cancer-associated tissues, the stromal microenvironment promotes prostate cell proliferation in the presence of DHEA via stromal metabolism to androgenic metabolites and induction of secondary growth factors. This notion is supported by literature demonstrating that the initial pre-neoplastic lesions of prostate cancer are associated with infiltration of immune cells and increased presence of cytokines. These studies will continue in exploring potential herbal-botanical agents, especially endocrine related, that may target these signaling proteins and modulate the response between the stromal and epithelial cells.
