Tumor-Derived Interleukin-8 Stimulates Osteolysis Independent of the Receptor Activator of Nuclear Factor-KB Ligand Pathway Manali S. Bendre, 1,2 Aaron G. Margulies, 3 Brandon Walser, 1,2 Nisreen S. Akel, 1,2 Sudeepa Bhattacharrya, 2 Robert A. Skinner, 2 Frances Swain, 2 Vishnu Ramani, 1,2 Khalid S. Mohammad, 4 Lisa L. Wessner, 4 Alfredo Martinez, 5 Theresa A. Guise, 4 John M. Chirgwin, 4 Dana Gaddy, 1,2 and Larry J. Suva 1,2 1 Department of Orthopaedic Surgery, Center for Orthopaedic Research, Barton Research Institute; 2 Department of Physiology and Biophysics; 3 Division of Breast Surgical Oncology, University of Arkansas for Medical Sciences, Little Rock, Arkansas; 4 Division of Endocrinology, Department of Medicine, University of Virginia, Charlottesville, Virginia; and 5 Department of Neuroanatomy and Cell Biology, Instituto Cajal, Consejo Superior de Investigaciones Cientificas, Madrid, Spain Abstract Bone is a common site of cancer metastasis. Breast, prostate, and lung cancers show a predilection to metastasize to bone. Recently, we reported that the chemokine interleukin 8 (IL-8) stimulates both human osteoclast formation and bone resorp- tion. IL-8 mRNA expression was surveyed in a panel of human breast cancer lines MDA-MET, MDA-MB-231, MDA-MB-435, MCF-7, T47D, and ZR-75, and the human lung adenocarcinoma cell line A549. IL-8 mRNA expression was higher in cell lines with higher osteolytic potential in vivo . Human osteoclast formation was increased by MDA-MET or A549 cell-conditioned medium, but not by MDA-MB-231. Pharmacologic doses of receptor activator of nuclear factor-KB (RANK)-Fc or osteo- protogerin had no effect on the pro-osteoclastogenic activity of the conditioned medium; however, osteoclast formation stimulated by conditioned medium was inhibited 60% by an IL-8-specific neutralizing antibody. The data support a model in which tumor cells cause osteolytic bone destruction independently of the RANK ligand (RANKL) pathway. Tumor- produced IL-8 is a major contributor to this process. The role of secreted IL-8 isoforms was examined by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, which detected distinct IL-8 isoforms secreted by MDA-MET and MDA-231 cells, suggesting different pro-osteoclastogenic activities of the two IL-8-derived peptides. These data indicate that ( a ) osteoclast formation induced by MDA-MET breast cancer cells and A549 adenocarcinoma cells is primarily mediated by IL-8, (b) cell-specific isoforms of IL-8 with distinct osteoclastogenic activities are produced by tumor cells, and (c ) tumor cells that support osteoclast formation independent of RANKL secrete other pro-osteoclastogenic factors in addition to IL-8. (Cancer Res 2005; 65(23): 11001-9) Introduction Bone is a common site of cancer metastasis. Several tumors show a particular predilection for metastasis to bone, including breast, prostate, lung, thyroid, and renal cancers (1). Of the 4 million people who die in the United States each year, approximately one quarter die from cancer and 70% of these have either breast, lung, or prostate cancer (1). Consequently, there are >350,000 people in the United States who die each year with bone metastases (1). Tumors cause two distinct but overlapping types of skeletal lesions when they spread to bone, either lytic or blastic, with many tumors demonstrating the pathologic features of both (1). After tumor cells find their way to the bone marrow, there are numerous cell types present in the microenvironment that are involved in the maintenance of immune and inflammatory responses and whose activity may be modulated by the cytokines and/or growth factors secreted by tumor cells (1). The progression of osteolytic bone metastases requires the establishment of functional interactions between metastatic cancer cells and bone cells (2), which are mediated by soluble regulators of osteoclast formation, activity, and survival (1). The number of osteoclasts is increased at metastatic sites in breast cancer patients (3). Tumor cells can interact with osteoblasts, which, in turn, stimulate osteoclasts to differentiate from hematopoietic precursors in the bone marrow, resulting in increased osteoclastic bone resorption (4). Cancer cells are known to produce a variety of stimulators of bone resorption, such as parathyroid hormone-related protein (PTHrP), interleukin 1 (IL-1), IL-6, IL-8, IL-11, colony-stimulating factor-1 (CSF-1), granulocyte macrophage-CSF, transforming growth factor (TGF)-h1, TGF-h2, tumor necrosis factor-a, insulin-like growth factor II, and hepatocyte growth factor (5–14). The secretion of some but not all of these factors by cancer cells regulates the expression of receptor activator of nuclear factor-nB ligand (RANKL) on the surface of stromal osteoblasts, thereby increasing osteoclast- mediated bone resorption (4, 5). Osteoclasts are also stimulated by tumor products (15, 16), but not usually by direct tumor cell secretion of RANKL (4). The bone marrow microenvironment is further enriched by growth factors released during osteoclastic bone resorption, which may support proliferation and growth of tumor cells or alter their phenotype in bone (17). In addition, tumor-derived products, such as dkk-1, can also inhibit osteoblast differentiation, thus also contributing to bone loss (18). Recently, we reported that the a chemokine IL-8 is a potent and direct activator of osteoclastic differentiation and bone resorption (2). The mechanism of action of this chemokine does not require activation of the RANKL pathway, but involves the expression and activation of the specific IL-8 receptor (CXCR1) on the surface of osteoclasts and their precursors (2). Note: A.G. Margulies is a Virginia Clinton Kelley/Fashion Footwear Association of New York Research Fellow in Diseases of the Breast and B. Walser was the recipient of a National Cancer Institute Partners in Research studentship at University of Arkansas for Medical Sciences. Requests for reprints: Larry J. Suva, Center for Orthopaedic Research, Department of Orthopaedic Surgery, Slot 644, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205. Phone: 501-526-6110; Fax: 501-686-8987; E-mail: suvalarryj@uams.edu. I2005 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-2630 www.aacrjournals.org 11001 Cancer Res 2005; 65: (23). 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