1860 Vol. 10, 1860–1869, March 1, 2004 Clinical Cancer Research Osteoblast-Derived Factors Induce Androgen-Independent Proliferation and Expression of Prostate-Specific Antigen in Human Prostate Cancer Cells Natalie Blaszczyk,1 Bassam A. Masri,4 cotreated with both OCM and androgen. OCM targeted the 1 1 Nasrin R. Mawji, Takeshi Ueda, NH2-terminal domain of the AR. The effect of OCM on transcriptional activity of the AR was inhibited by an anti- Gavan McAlinden,4 Clive P. Duncan,4 1 2 androgen. Neutralizing antibodies to IL-6 blocked prolifer- Nicholas Bruchovsky, Hans-Udo Schweikert, ation and expression of PSA by OCM. 2 3 Doris Schnabel, Edward C. Jones, and Conclusion: Osteoblasts secrete factors, such as IL-6, Marianne D. Sadar1 that cause androgen-independent induction of PSA gene 1Department of Cancer Endocrinology, British Columbia Cancer expression and proliferation of prostate cancer cells by a Agency, Vancouver, British Columbia, Canada; 2Department of mechanism that partially relies on the AR. Identifying such Internal Medicine, University of Bonn, Bonn, Germany; and molecular mechanisms may lead to improved clinical man- 3 4 Anatomical Pathology and Orthopaedic Surgery, Vancouver agement of metastatic prostate cancer. General Hospital, British Columbia, Canada INTRODUCTION ABSTRACT Localized carcinoma of the prostate (CaP) can potentially Purpose: Prostate cancer metastasizes to the skeleton to be cured by surgery or radiation therapy. However, the only form osteoblastic lesions. Androgen ablation is the current treatment available for advanced metastatic disease is the with- treatment for metastatic prostate cancer. This therapy drawal of androgens, which are essential for the survival of is palliative, and the disease will return in an androgen- prostate epithelial cells (for review, see Ref. 1). Clinical re- independent form that is preceded by a rising titer of pros- sponse to androgen ablation therapy is temporary with the tate-specific antigen (PSA). Here, we investigated the possi- ultimate development of androgen-independent disease that is bility that human osteoblasts might secrete factors that preceded by an increasing titer of serum prostate-specific anti- contribute to the emergence of androgen-independent pros- gen (PSA). Unlike most malignancies, up to 85% of CaP me- tate cancer. tastases occur in the bone as osteoblastic (bone forming) lesions. Experimental Design: Primary cultures of human osteo- Osteoblastic lesions are the major cause of CaP-related morbid- blasts were used as a source of conditioned medium (OCM). ity, and mortality and even small, bone-limited tumor burden in Proliferation, expression of androgen-regulated genes, and patients are strongly correlated to cachexia and death (for re- transactivation of the androgen receptor (AR) were moni- view, see Ref. 2). tored in LNCaP human prostate cancer cells in response to Bone is mostly composed of an acellular collagen matrix and OCM using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- abundant in immobilized growth factors. Dispersed throughout this tetrazolium bromide (MTT) assay, Northern blot analysis, matrix are osteoblasts and osteoclasts, which are the cells respon- and reporter gene constructs. Levels of interleukin-6 (IL-6) sible for bone maintenance. In bone, remodeling osteoclasts de- present in OCM were measured, and its contribution to grade the matrix to release growth factors which stimulate osteo- proliferation and expression of PSA were investigated by blasts to lay down new bone (for review, see Refs. 3 and 4). Many neutralization studies with anti IL-6 antibodies. of these growth factors increase proliferation of prostate cancer Results: OCM increased the proliferation and expres- cells. In vitro studies have shown increased proliferation of prostate sion of PSA at both the protein and RNA levels in LNCaP cancer cells stimulated by conditioned media from or cocultured cells. Synergistic increases in the activities of PSA (6.1 kb)- with osteoblasts, growth factor extracts, and individual growth and pARR3-tk-luciferase reporters were measured in cells factors derived from bone, such as interleukin-6 (IL-6), insulin-like growth factor I (IGF-I) and IGF-II, epidermal growth factor, kera- tinocyte growth factor, bone morphogenic proteins, fibroblast growth factors, transforming growth factor-␤, cytokines, platelet- derived growth factor, vascular endothelial growth factor, and Received 6/17/03; revised 11/19/03; accepted 12/1/03. endothelin-1 (5–13). Many of these growth factors may be in- Grant support: George M. O’Brien Research Center Grant P50 volved in circumventing the need for androgen in advanced disease DK47656 from the NIH (to M. D. S.). The costs of publication of this article were defrayed in part by the by a mechanism involving ligand-independent activation of the payment of page charges. This article must therefore be hereby marked androgen receptor (AR; Refs. 6 and 14–17). advertisement in accordance with 18 U.S.C. Section 1734 solely to The AR is a transcription factor that binds androgens and indicate this fact. regulates gene expression required for normal male sexual de- Requests for reprints: Marianne D. Sadar, Department of Cancer Endocrinology, British Columbia Cancer Agency, 600 West 10th Ave- velopment and maintenance of secondary sex characteristics nue, Vancouver, B. C., V5Z 4E6 Canada. Phone: (604) 877-6036; Fax: (for review, see Ref. 1). It is expressed in the majority of (604) 877-6011; E-mail: [email protected]. prostate cancer tissue specimens, including androgen independ- Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2004 American Association for Cancer Research. Clinical Cancer Research 1861 ent or hormone refractory disease, and is therefore a strong All experiments were performed using serum- and phenol red- candidate for mediating androgen resistance (18–20). The AR is free conditions. composed of three domains. Centrally located is the DNA- Culturing of Osteoblast-Like Cells and Preparing binding domain (DBD) that binds to androgen response ele- Osteoblast-Conditioned Medium (OCM)-MEM. Human ments (AREs) in upstream regulatory regions of androgen- osteoblast-like cells were cultured from femoral head trabecular regulated genes, such as PSA. The most COOH-terminal region explants obtained from osteoarthritis patients undergoing hip/ comprises the ligand-binding domain that binds androgens and knee replacement surgery. To minimize possible patient varia- antiandrogens, such as bicalutamide. The ligand-binding do- tion, donors were restricted to males Ͻ 65 years of age. Tra- main contains a weak activation function-2 region and is sepa- becular bone was scraped into bone chips and further processed rated from the DBD by a hinge region, which mediates nuclear with a mortar and pestle. Bone chips were cultured in MEM containing 20% FBS at 37°C in the presence of 5% CO (35). localization. The NH2-terminal domain (NTD) is the most vari- 2 able in sequence homology between species and contains the The outgrowth of the osteoblast-like cells was monitored visu- activation function-1 region required for transactivation (for ally under the microscope with the aid of Gram Safaran staining. review, see Ref. 1). The AR can be activated in an androgen- Cells were characterized using the reverse transcriptase-PCR for independent manner by a number of factors, including IL-6, IGF expression of the following osteoblast markers: (a) type I pro- I, IGF II, keratinocyte growth factor, epidermal growth factor, collagen; (b) alkaline phosphatase; and (c) osteocalcin (35). forskolin, and cAMP, and some factors such as IL-6 and cAMP When the osteoblast-like cells were confluent, they were washed ϫ have been shown to mediate activation via the AR NTD (6, with PBS (3 20 ml) and cultured in 20 ml of serum-free MEM. After 48 h, the OCM was collected and centrifuged or 14–16). Because the bone environment is rich in many of these filtered through 0.22-␮m Nalgene units to remove the cellular growth factors, it has been suggested that bone-derived factors debris before storing at Ϫ80°C until use. OCMs from primary may facilitate survival and progression of CaP to androgen cultures of osteoblasts prepared from different patients were independence by cross-talk with the AR and alternative signal never pooled but rather used individually. Primary cultures of transduction pathways (5, 8, 21). osteoblasts were grown and maintained in Falcon Primaria T75 Clinical and experimental evidence suggests that one of the flasks, with a surface of 75 cm2. The average protein concen- more important factors in prostate cancer progression to andro- tration in OCM was 0.28 Ϯ 0.05 mg/ml (SE). Fibroblast- gen independence and development of osseous metastases is conditioned media were collected and used as reported pre- IL-6. Elevated levels of IL-6 have been found in sera from viously (5). patients who have metastatic and/or hormone refractory disease 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (22–26). IL-6 was first characterized as a modulator of an Bromide (MTT) Assay. LNCaP cells (1 ϫ 104) were plated immune response that is produced by T cells and involved in in 96-well Falcon tissue culture plates in RPMI containing 0.5% hematopoiesis and inflammation (for review, see Ref. 27). How- FBS in a final volume of 0.1 ml. When the cells reached 60% ever, IL-6 has been subsequently shown to be produced by many confluence, usually within 24 h, they were treated with R1881 other cell types, including normal and malignant prostate epi- or OCM collected from preparations of osteoblasts from three thelial cells and bone cells (28–34). It is clear that IL-6 has the individual patients. After 5 days of culture, cell proliferation potential to act in both a paracrine and autocrine manner. Im- was assessed by adding 50 ␮l of 3-(4,5-dimethylthiazol-2-yl)- portantly, IL-6 has been shown to enhance proliferation of 2,5-diphenyltetrazolium bromide dye (1 mg/ml) in serum-free prostate cancer cells, including LNCaP (6, 28), and induce media to the cells.