[CANCER RESEARCH 64, 7156 –7168, October 1, 2004] Changes in Androgen Receptor Nongenotropic Signaling Correlate with Transition of LNCaP Cells to Androgen Independence Emmanual Unni, 1,3 Shihua Sun, 1,3 Bicheng Nan, 1,3 Michael J. McPhaul, 4 Boris Cheskis, 5 Michael A. Mancini, 2 and Marco Marcelli 1,2,3 Departments of 1 Medicine and 2 Molecular and Cellular Biology, 3 Baylor College of Medicine and VA Medical Center, Houston, Texas; 4 Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas; and 5 Department of Cell Biology, Women’s Health Research Institute, Wyeth Pharmaceuticals, Collegeville, Pennsylvania ABSTRACT A cure for prostate cancer (CaP) will be possible only after a complete understanding of the mechanisms causing this disease to progress from androgen dependence to androgen independence. To carry on a careful characterization of the phenotypes of CaP cell lines before and after acquisition of androgen independence, we used two human CaP LNCaP sublines: LNCaP nan , which is androgen dependent (AD), and LNCaP-HP, which is androgen independent (AI). In AD LNCaP nan cells, dihydrotes- tosterone (DHT) stimulated in an androgen receptor (AR)-dependent way a phosphorylation signaling pathway involving steroid receptor coactiva- tor (Src)–mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)-1/2–ERK-1/2– cAMP-response element binding- protein (CREB). Activation of this pathway was associated with increased [ 3 H]thymidine incorporation and resistance to apoptosis. Use of domi- nant-negative forms of MEK-1/2 and CREB demonstrated in LNCaP nan cells that DHT induced [ 3 H]thymidiine incorporation through a thus far unidentified molecule activated downstream of MEK-1/2, and antiapopto- sis through phosphorylation of the transcription factor CREB. In con- trast, in AI LNCaP-HP cells, the Src–MEK-1/2–ERK-1/2–CREB pathway was constitutively active. Because it was not further stimulated by addi- tion of DHT, no increase of [ 3 H]thymidine incorporation or apoptosis resistance was demonstrated in LNCaP-HP cells. Additional experiments showed that Src and the scaffold protein MNAR coimmunoprecipitated with AR, indicating a role for Src as an apical molecule in the Src–MEK- 1/2–ERK-1/2–CREB pathway. Interestingly, differences between the two cell lines were that in LNCaP-HP cells presence of an AI phenotype and lack of response to DHT were associated with constitutive activation of the protein kinase Src and interaction among Src, AR, and MNAR. In con- trast, in LNCaP nan cells, presence of an AD phenotype and ability to respond to DHT were associated with DHT-dependent activation of Src kinase activity and interaction among Src, AR, and MNAR. Intriguingly, in LNCaP nan cells, we found that transcription through the prototypical CREB-responsive promoter c-fos could be induced in a DHT-dependent way, and this action was inhibited by the AR antagonist Casodex and MEK-1 inhibitor PD98059. In contrast, transcription through the PSA P/E promoter, a prototypical AR-dependent promoter directly activated by agonist, was obliterated only by Casodex. Additional experiments with genital skin fibroblasts derived from patients with a variety of AR abnor- malities indicated that nongenotropic AR signaling does not depend on an intact DNA-binding domain or on the ability of AR to translocate to the nucleus. The results suggest the following: (1) Constitutive activation of the Src–MEK-1/2–ERK-1/2–CREB pathway is associated with the AI phenotype observed in LNCaP-HP cells. (2) Activation of the Src–MEK- 1/2–ERK-1/2–CREB pathway is DHT dependent in AD LNCaP nan cells. (3) DHT activation of this pathway is associated with induction of [ 3 H]- thymidine incorporation by a molecule activated downstream of MEK-1/2 and of antiapoptosis through activation of the transcription factor CREB in AD LNCaP nan cells. (4) AR regulates transcription either directly upon ligand binding and nuclear translocation or indirectly through kinase pathways leading to activation of downstream transcription factors. (5) Nuclear translocation and ability of the DNA-binding domain of AR to interact with DNA are not prerequisites for nongenotropic AR activity. INTRODUCTION As a member of the superfamily of nuclear receptors, androgen receptor (AR) is a ligand-dependent transcription factor controlling the expression of specific genes (1). At least 60 minutes are necessary for AR to alter gene expression (2). Primarily, positive or negative AR-modulated gene transcription involves an initial phase of ligand binding to the receptor in the cytoplasm, followed by nuclear trans- location, contact with the promoter of AR-dependent genes, and the general transcription machinery through a number of coactivators or corepressors. However, increasing evidence indicates that signal transduction pathways known to trigger cell proliferation are activated by steroid receptors by mechanisms other than direct receptor mod- ulation of gene expression (2). This alternative mechanism of action has been dubbed “rapid” or “nongenomic” signaling and in some cases is measurable within seconds after the addition of ligand to cultured cells (3). Rapid signaling by steroid hormone receptors was identified 37 years ago and has now been reported for nearly all nuclear receptor family members (2, 4), including glucocorticoid (5), mineralocorticoid (6), estrogen (ER and ER; refs. 7 and 8), pro- gesterone (9, 10), and ARs (11–15). With the discovery that steroid receptors prevent bone loss in mice (16) and induce Xenopus oocyte maturation (14) by using a rapid signaling mechanism, these alterna- tive pathways of steroid hormone action have been firmly established as having important physiologic roles. With the recent identification of a membrane progesterone receptor with the structure of a G protein (17, 18), the potential importance of nongenotropic signaling of steroid receptors has increased even further. Several breakthroughs have revealed important new insights into the mechanism of AR action, and much of this new knowledge has been applied to the field of prostate cancer (CaP) development and progression. The prostate is a quintessential AR-dependent organ. Clearly, the presence of a functional AR and of normal levels of agonists are conditio sine qua for prostate development to the normal adult phenotype (19, 20). Maintenance of the prostate during adult- hood also depends on the presence of physiologic concentrations of these ligands, and castration is associated with abrupt prostate invo- lution (apoptosis; ref. 21). AR signaling plays at least a permissive role in the development of CaP. For instance, some animal models of CaP require normal level of androgens (endogenous or exogenous) for CaP to develop (22), whereas the lack of androgens observed in the eunuch population of China was associated with a lack of CaP development (23). Further- more, a positive correlation exists between the development of CaP and AR activity. For example, increased AR expression and signaling in the prostate are associated with increased precancerous lesions in a transgenic model (24). In vitro experiments have shown that a short- Received 3/29/04; revised 6/29/04; accepted 8/3/04. Grant support: Department of Defense Prostate Cancer Research grant DAMD17- 00-1-0075, the Merit Review Program of the Veterans Administration (M. Marcelli), NIH grants DK 55622 (M. Mancini) and DK03892, and Robert A. Welch grant I-1090 (M. McPhaul). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Marco Marcelli, 2002 Holcombe Boulevard, Houston, TX 77030. Phone: 713-794-7945; Fax: 713-794-7714; E-mail: marcelli@bcm.tmc.edu. ©2004 American Association for Cancer Research. 7156 Downloaded from http://aacrjournals.org/cancerres/article-pdf/64/19/7156/2518495/zch01904007156.pdf by guest on 11 July 2022