Prostate Cancer Cells-Osteoblast Interaction Shifts Expression of Growth/Survival-related Genes in Prostate Cancer and Reduces Expression of Osteoprotegerin in Osteoblasts 1 Karim Fizazi, 2 Jun Yang, 2 Sara Peleg, Charles R. Sikes, Erica L. Kreimann, Danai Daliani, Matilde Olive, Kevin A. Raymond, Todd J. Janus, Christopher J. Logothetis, Gerard Karsenty, and Nora M. Navone 3 Departments of Genitourinary Medical Oncology [K. F., J. Y., C. R. S., E. L. K., D. D., M. O., C. J. L., N. M. N.], Endocrine Neoplasia and Hormonal Disorders [S. P.], and Pathology [K. A. R.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030; Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030 [G. K.]; and Abbott Laboratories, Abbott Park, Illinois [T. J. J.] ABSTRACT Purpose: Prostate cancer specifically metastasizes to bone where it leads to bone formation. We previously reported that coculturing MDA PCa 2b prostate cancer cells with primary mouse osteoblasts (PMOs) induced PMO proliferation and differentiation. An osteoblastic reaction was also observed in vivo after injection of MDA PCa 2b cells into the bones of severe combined immuno- deficient disease mice. The aim of this study was to iden- tify the sequence of events that leads to these osteoblastic lesions in vivo and, using this in vitro model, to define the contributions of various genes and cellular pathways in the pathophysiology of osteoblastic bone metastases of prostate cancer. Experimental Design and Results: We show histological evidence of de novo bone formation as early as 2 weeks after injection of MDA PCa 2b cells in the bone of severe combined immunodeficient disease mice. In vitro, we show that PMOs induce MDA PCa 2b proliferation, suggesting a synergistic paracrine loop between these cells and PMOs. Endothelin (ET)-1, which is a mitogen for several cell types, is produced by all prostate cancer cell lines tested, and Atrasentan, an antagonist of ET-1 receptor A, partially reversed PMO proliferation induced by MDA PCa 2b cells. ET-1 is known to be comitogenic with a number of growth factors, including insulin-like growth factor (IGF)-I. In this study, we report that IGF-binding protein (IGFBP)-3 transcripts (that regulate levels of free IGF) are down-regulated in prostate cancer cells co- cultured with PMO, whereas prostate-specific antigen (a protease known to cleave IGFBP-3) is detected in the 150 – 400 ng/ml range. Accordingly, IGFBP-3 has antipro- liferative effects in PMOs, which were attenuated in our in vitro system. Taken together, our studies also implicate the IGF axis to play a role in this model of bone metas- tases. Secondly, the transcript level of mouse double minute 2 (a protein that regulate p53) was increased in prostate cancer cells grown with PMOs. The p53- dependent and -independent oncogenic activities of mouse double minute 2 suggest that osteoblasts induce a survival advantage in prostate cancer cells. Lastly, we show that expression of osteoprotegerin is decreased and of receptor activator of nuclear factor-B ligand is increased in PMOs cultured in the presence of MDA PCa 2b cells, two events associated with osteoclast activation and bone resorption. Conclusions: Our results provide evidence that mul- tiple and distinct molecular events affecting both bone formation and bone resorption concur to the increase bone mass in prostate cancer bone metastases. These data also provide a rationale for developing therapeutic strat- egies designed to target these molecular changes. INTRODUCTION Prostate cancer is the most frequent form of cancer in men in the United States (1). It is associated with high morbidity and mortality rates as a result of its tendency to metastasize to the bone (2). Bone is usually the first and exclusive site of progression of the disease to the androgen- independent stage, and the bone metastases are typically osteoblastic (i.e., involving excessive bone formation by os- teoblasts; Refs. 2– 4). These observations suggest that pros- tate cancer cells stimulate cells of osteoblast lineage at this site of metastasis and that the prostate cancer cell-osteoblast interaction contributes to the lethal progression of prostate cancer. The molecular bases for this interaction are poorly understood, partially because few in vivo models mimic the natural progression and dissemination of prostate cancer (5, 6). Moreover, even fewer systems lead to osteoblastic le- sions, the most frequent phenotype of bone metastases from prostate cancer in humans. A number of soluble factors, Received 10/3/02; revised 1/6/03; accepted 2/14/03. 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 This work was supported by Association for the Cure of Cancer of the Prostate, DAMD17-991-9028, NIH Grants CA75499-04, DK50583, AR45548, and the Ligue contre le cancer. 2 K. F. and J. Y. contributed equally to this work. 3 To whom requests for reprints should be addressed, at Department of GU Medical Oncology, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 013, Houston, TX 77030. Phone: (713) 792-2898; Fax: (713) 792-5696; E-mail: nnavone@mail.mdanderson.org. 2587 Vol. 9, 2587–2597, July 2003 Clinical Cancer Research Cancer Research. on December 12, 2021. © 2003 American Association for clincancerres.aacrjournals.org Downloaded from