[CANCER RESEARCH 62, 1549 –1554, March 1, 2002] Activation of the Erk Mitogen-activated Protein Kinase Pathway Stimulates Neuroendocrine Differentiation in LNCaP Cells Independently of Cell Cycle Withdrawal and STAT3 Phosphorylation 1 Jayoung Kim, Rosalyn M. Adam, and Michael R. Freeman 2 The Urologic Laboratory, Department of Urology, Children’s Hospital, and the Department of Surgery, Harvard Medical School, Boston, Massachusetts 02115 ABSTRACT Neuroendocrine (NE) differentiation in prostate cancer (PCa) has been found in some studies to correlate with unfavorable clinical outcome. The mechanisms by which PCa acquires NE properties are poorly understood. In this study, we demonstrate that heparin-binding epidermal growth factor-like growth factor (HB-EGF), a prostate smooth muscle-derived mitogen and survival factor, can evoke NE differentiation in LNCaP human PCa cells. HB-EGF induction of NE differentiation was mediated by a mitogen-activated protein kinase (MAPK) kinase-dependent mecha- nism, and this process was blocked by p38 MAPK signaling. NE differ- entiation induced by HB-EGF occurred independently of STAT3 phos- phorylation and coincided with continued cell cycle transit. These findings suggest that endogenous stroma-derived factors, acting through MAPK signaling pathways, may play a significant role in the acquisition of NE properties by PCa cells. They also demonstrate that withdrawal from the cell cycle is not a prerequisite for expression of NE characteristics by PCa. INTRODUCTION Epithelial cells of the human prostate are generally classified as either basal, secretory, or NE, 3 based on their morphology, location, and expression of marker proteins. NE cells comprise a minor sub- population of the normal prostatic epithelium, and PCa exhibiting predominantly NE characteristics are rare, comprising only 0.5–2% of clinical specimens. Nevertheless, NE differentiation in PCa is of interest because tumors with a prominent NE component are typically androgen independent and highly aggressive and because many PCa are infiltrated with NE-like cells (1–3). Although reports vary on the extent of NE differentiation in PCa (from 5 to 80%), evaluation of NE cell number has been correlated with androgen-independent disease and poor prognosis in some studies (4 –7). Several authors have argued that even a small NE component has the potential to stimulate growth and progression of PCa, primarily through paracrine mecha- nisms. NE-like cells can secrete a variety of cytokines and neurohormones linked to growth and survival in prostate and other cancer cells (8). Secretion of these molecules into the extracellular space by NE cells may affect adjacent adenocarcinoma (9 –13). Furthermore, the NE differentiation process itself has been proposed to involve cell cycle arrest (14, 15), thus rendering differentiated tumor cells resistant to radiation and chemotherapy. The molecular basis by which NE cells arise in prostatic tumors is not understood; however, studies have demonstrated that significant plasticity exists between the adenocarcinoma and NE phenotypes. This suggests that NE cells arise from adenocarcinoma cells in re- sponse to epigenetic stimuli. NE differentiation can be induced in PCa cells by several means, including IL-6, agents that increase levels of intracellular cyclic AMP, and culture in steroid-depleted medium (14 –19). Although the transcription factor, STAT3, and the tyrosine kinase, Etk, have been implicated in NE differentiation in LNCaP human PCa cells (17, 20, 21), the signaling mechanisms underlying acquisition of NE properties by PCa cells remain undefined. More- over, the potential role of the prostatic microenvironment, e.g., the influence of the prostatic stroma, in control of NE differentiation in PCa has not been established. HB-EGF is an EGFR/ErbB1 ligand that is synthesized primarily by interstitial and vascular SMCs of the prostatic stroma (22). HB-EGF is a potent mitogen and survival factor for prostate epithelial and carcinoma cells (22–24). EGFR activation by HB-EGF and other ErbB1 ligands triggers signaling through the Erk MAPK pathway, resulting in diverse biological responses (25). The stromal location of HB-EGF synthesis, coupled with the observation that EGFR is ex- pressed principally by prostatic epithelial and carcinoma cells (24), suggests that HB-EGF mediates directional paracrine signaling from the SMCs to the epithelial compartment. In this study, we demonstrate that HB-EGF is a mediator of NE differentiation in PCa cells. Our results suggest that endogenous stroma-derived factors, acting through the Erk-MAPK signaling path- way, may play a significant role in the acquisition of NE properties by PCa cells. MATERIALS AND METHODS Reagents. Human recombinant HB-EGF and neutralizing anti-HB-EGF antibody were purchased from R&D Systems, Inc. (Minneapolis, MN). Anti- NSE antibody was obtained from Neomarkers (Fremont, CA). IL-6 was from Upstate Biotechnology, Inc. (Lake Placid, NY), and charcoal dextran-treated serum was from Hyclone Laboratories, Inc. (Logan, UT). SB203580, PD153035, and PD098059 were from Calbiochem (San Diego, CA). Antiphos- pho-Erk, anti-Erk, antiphospho-p38, anti-p38, antiphospho-STAT3 (Tyr705), antiphospho-STAT3 (Ser727), and anti-STAT3 antibodies were purchased from Cell Signaling Technology (Beverly, MA). Antiactin antibody was from Sigma Chemical Co. (St. Louis, MO). Cultured Cells. The human PCa cell line LNCaP was purchased from American Type Culture Collection (Rockville, MD) and cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, 100 units/ml penicillin, and 100 g/ml streptomycin (Life Technologies, Inc., Grand Island, NY). These supplements were used in all of the media unless otherwise indicated. Cells were cultured in a humidified atmosphere of 5% CO 2 at 37°C. HB-EGF Expression Construct and Generation of Stable Transfec- tants. To generate sHB-EGF, a fragment encoding the mature soluble form of HB-EGF was amplified by PCR from a previously engineered HB-EGF construct (26) using the primers: 5'-GGATCCATGAAGCTGCTGCCGTCG- GTGGTG-3' and 5'AAGTCTGGGCCCTTCCACTGGGAGGCTCAG-3', Received 6/20/01; accepted 1/16/02. 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. 1 Supported by NIH Grants R37 DK47556, RO1 CA77386, and RO1 DK57691 (to M. R. F.). R. M. A. is an American Foundation for Urological Disease Research Scholar. 2 To whom requests for reprints should be addressed, at John F. Enders Research Laboratories, Room 1161, Children’s Hospital Boston, 300 Longwood Avenue, Boston, MA 02115. Phone: (617) 355-6054; Fax: (617) 355-7760; E-mail: michael.freeman@ tch.harvard.edu. 3 The abbreviations used are: NE, neuroendocrine; PCa, prostatic adenocarcinoma; Erk, extracellular signal regulated; EGFR, epidermal growth factor receptor; HB-EGF, heparin-binding EGF-like growth factor; MAPK, mitogen-activated protein kinase; SMC, smooth muscle cell; FBS, fetal bovine serum; sHB-EGF, constitutively secreted mutant of HB-EGF; NSE, neuron-specific enolase; cFBS, charcoal-stripped fetal bovine serum; STAT3, signal transducers and activators of transcription 3; MKK3b(EE), a constitutively activated mutant of MKK3b; MEK, mitogen-activated protein/extracellular signal- regulated kinase kinase; IL, interleukin; PI3k, phosphatidylinositol 3'-kinase. 1549 Research. on November 5, 2015. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from