Human prostate stromal cells stimulate increased PSA production in DHEA-treated prostate cancer epithelial cells

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Abstract

Dehydroepiandrosterone (DHEA) is commonly used as a dietary supplement and may affect prostate pathophysiology when metabolized to androgens and/or estrogens. Human prostate LAPC-4 cancer cells with a wild type androgen receptor (AR) were treated with DHEA, androgens dihydrotestosterone (DHT), T, or R1881), and E2 and assayed for prostate specific antigen (PSA) protein and gene expression. In LAPC-4 monocultures, DHEA and E2 induced little or no increase in PSA protein or mRNA expression compared to androgen-treated cells. When prostate cancer-associated (6S) stromal cells were added in coculture, DHEA stimulated LAPC-4 cell PSA protein secretion to levels approaching induction by DHT. Also, DHEA induced 15-fold more PSA mRNA in LAPC-4 cocultures than in monocultures. LAPC-4 proliferation was increased 2–3-fold when cocultured with 6S stromal cells regardless of hormone treatment. DHEA-treated 6S stromal cells exhibited a dose- and time-dependent increase in T secretion, demonstrating stromal cell metabolism of DHEA to T. Coculture with non-cancerous stroma did not induce LAPC-4 PSA production, suggesting a differential modulation of DHEA effect in a cancer-associated prostate stromal environment. This coculture model provides a research approach to reveal detailed endocrine, intracrine, and paracrine signaling between stromal and epithelial cells that regulate tissue homeostasis within the prostate, and the role of the tumor microenvironment in cancer progression.

Introduction

Dehydroepiandrosterone (DHEA), the most abundant endogenous adrenal steroid produced by men and women, declines markedly with aging [1]. It is increasingly consumed as an over-the-counter dietary supplement for its purported antiaging effects, yet its usefulness, long-term safety and effects on prostate tissues remain uncertain [2]. Prostate stromal and epithelial cells possess the enzymatic machinery to metabolize DHEA to more active androgenic and/or estrogenic steroids [3], [4], [5] (intracrine) and express secondary mediators (paracrine) for epithelial growth and differentiation. Defining DHEA and other steroid hormonal responses in the prostate requires simulation of the intracrine and paracrine effects of hormones provided by stromal cells. Interactions between stromal and epithelial cells in hormone responsive tissues are increasingly recognized as an important area of research highlighted for many diseases, especially cancer [6].

As a precursor to both estrogens and testosterone, DHEA excess may pose a potential cancer risk in hormone-responsive tissues such as the prostate. Different prostate cell types display distinct responses to DHEA in vitro. For example, human prostate cancer LNCaP cells, which harbor a mutated androgen receptor (AR), respond to DHEA administration with increased proliferation, and expression of prostate specific antigen (PSA) and IGF axis proteins [7]. Human prostatic primary stromal cells respond to androgens, such as dihydrotestosterone (DHT), but not DHEA, by increasing IGF-I secretion [8]. Contrastingly, in in vivo studies, DHEA has been found to be an effective inhibitor to carcinogen-induced prostate cancers [9]. In the current study, we examined the effects of DHEA on proliferation and PSA production in human prostate cancer LAPC-4 cells, which contain a normal AR, in both monoculture and in coculture with stromal cells from normal or cancer-related tissues. This cell coculture model provides an opportunity to investigate the intracrine and paracrine stromal mediators of hormonal responses in epithelial cells and demonstrates the importance of prostate stromal cells in mediating the response of prostate epithelial cells to DHEA.

Section snippets

Cell culture

LAPC-4 cells were derived from lymph node metastases of a patient with advanced prostate cancer [10] and were generously provided by Dr. Charles Sawyers, UCLA. Primary human prostate cancer-derived stromal cells were isolated from radical prostatectomy specimens ([11]; cell lots from different patient samples were labeled: 5S, 6S, 9S, and 12S; kindly provided by Dr. John Isaacs, Johns Hopkins School of Medicine) and have been previously described [8]. Primary prostate stroma cells (PRSC)

DHEA effects on LAPC-4 PSA protein expression in monoculture

PSA secretion or expression was used as a biomarker for prostate epithelial cell response to androgenic hormones. LAPC-4 cells were assayed for PSA protein expression by ELISA in response to DHEA, compared to DHT, T and E2. The androgens, DHT and T induced PSA production in a dose-dependent manner with the maximal effect of 22 ng/mL per 100,000 cells at 10 nM DHT and of 19 ng/mL per 100,000 cells at 100 nM T (Fig. 1A, P < 0.0001). DHEA and E2 induction of PSA were minimal and only at 1000 nM (0.88 and

Discussion

This study provides a physiologically relevant in vitro endocrine (DHEA)–paracrine (stroma-mediated) reproduction of human prostate stromal cell induction of proliferation and PSA production in epithelial cancer cells. The data also support a mechanism of DHEA effects in prostate that would not be ascertained using single cell cultures alone. DHEA treatment did not affect prostate epithelial cells PSA production when cultured alone but produced an androgenic response in epithelial cells in the

Acknowledgements

The authors thank Dr. Charles Sawyers (UCLA) for providing the LAPC-4 cells, Dr. John Isaacs (Johns Hopkins School of Medicine) for providing the primary prostate stromal cells (5S, 6S, 9S, 12S) and Dr. Vernon Steele (NIH-NCI) and Dr. Angela Brodie (University of Maryland) for their constructive comments upon reviewing this manuscript. This work was supported by the Intramural Research Program, National Center for Complementary and Alternative Medicine, National Institutes of Health, Bethesda,

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