The Activity of Zoledronic Acid on Neuroblastoma Bone Metastasis Involves Inhibition of Osteoclasts and Tumor Cell Survival and Proliferation Hongjun Peng, 1,5 Yasuyoshi Sohara, 1,5 Rex A. Moats, 2,5 Marvin D. Nelson, Jr., 2,5 Susan G. Groshen, 3 Wei Ye, 3 C. Patrick Reynolds, 1,5 and Yves A. DeClerck 1,4,5 1 Division of Hematology-Oncology, Department of Pediatrics and Departments of 2 Radiology, 3 Preventive Medicine, and 4 Biochemistry and Molecular Biology, University of Southern California; 5 The Saban Research Institute of Children’s Hospital Los Angeles, Los Angeles, California Abstract Metastasis to the bone is seen in 56% of patients with neuroblastoma and contributes to morbidity and mortality. Using a murine model of bone invasion, we have reported previously that neuroblastoma cells invade the bone by activating osteoclasts. Here, we investigated the antitumoral and antiosteolytic activities of zoledronic acid, a bisphospho- nate inhibitor of osteoclasts, in combination with cytotoxic chemotherapy in our model. We first show that zoledronic acid given at the same time (early prevention) or 2 weeks after tumor cell injection (late prevention) significantly prevented the formation of severe osteolytic lesions. It also prevented formation of these lesions when given 4 weeks after tumor cell injection (intervention) when combined with chemotherapy including cyclophosphamide and topotecan. The combination of zoledronic acid + cyclophosphamide/topotecan also signi- ficantly improved survival (P < 0.001). In mice treated with zoledronic acid, we observed a marked inhibition of osteo- clasts inside the bone associated with a decrease in tumor cell proliferation and increase in tumor cell apoptosis. In vitro , zoledronic acid inhibited neuroblastoma cell proliferation and induced apoptosis, and these effects were significantly enhanced by the addition of 4-hydroxyperoxycyclophospha- mide (4-HC). The proapoptotic effect of zoledronic acid and zoledronic acid in combination with 4-HC on tumor cells was associated with an increase in caspase-3 activity and a decrease in phosphorylated Bcl-2, Bcl-2, and Bcl-X L expres- sion. Zoledronic acid inhibited the association of Ras with the plasma membrane and activation of c-Raf, Akt, and extracel- lular signal-regulated kinase 1/2. The data indicate that zoledronic acid, in addition to inhibiting osteoclasts, is active against tumor cells and suggest that zoledronic acid in combination with cytotoxic chemotherapy may be effective in children with neuroblastoma that has metastasized to the bone. [Cancer Res 2007;67(19):9346–55] Introduction Neuroblastoma is a tumor derived from the neural crest and the most common extracranial solid neoplasm in children (1). Although the event-free survival of patients with nonmetastatic and noninvasive tumors (stage I or II) is in the range of 90% to 95%, patients with high-risk disease have only a 3-year event-free survival of 42% when treated with intensive myeloablative chemotherapy, radiotherapy, autologous bone marrow transplan- tation, and 13-cis -retinoic acid (2). Metastasis at diagnosis is common in patients with neuroblastoma and sites most frequently involved include bone marrow, bone, lymph nodes, liver, and intracranial and orbital sites. Neuroblastoma rarely metastasizes to the lungs or the brain (3). The mechanisms involved in the formation of bone metastasis in neuroblastoma have now begun to be elucidated. It is documented that, as observed in breast cancer and multiple myeloma, neuroblastoma bone metastases are predominantly osteolytic (4, 5). The formation of osteolytic lesions by metastatic neuroblas- toma cells involve the recruitment and activation of osteoclasts, which can occur through two mechanisms (6). A first mechanism consists of the direct activation of osteoclasts by neuroblastoma cells. For example, when cultured in the presence of murine bone marrow cells, neuroblastoma cells express the receptor activator of nuclear factor-nB ligand (RANKL), a potent stimulator of RANK- expressing osteoclasts. This effect is abrogated in the presence of the RANKL inhibitor, osteoprotegerin (soluble RANK or OPG; refs. 7, 8). This mechanism of osteoclast activation is used by breast cancer cells as they metastasize to the bone (9, 10). A second mechanism reported recently by our laboratory involves the contribution of bone marrow–mesenchymal stem cells (BM- MSC), which in the presence of neuroblastoma cells express interleukin-6 (IL-6), a potent stimulator of osteoclasts (11). This mechanism was also reported in myeloma cells, which on contact with bone marrow stromal cells stimulate IL-6 expression (12), but in neuroblastoma, stimulation of IL-6 expression does not require cell-cell contact. At the convergence of these two mechanisms are the activation of osteoclasts and the degradation of mineralized bone (13). Bisphosphonates are a family of synthetic compounds, whose structure is derived from pyrophosphoric acid, which binds with high affinity to the bone matrix (14). These compounds are potent inhibitors of osteoclast activity that are currently used in the clinic for a variety of benign and malignant conditions associated with increased bone resorption, such as postmenopausal osteoporosis (15), malignant hypercalcemia (16), and bone metastasis (17). Bisphosphonates have been proposed as potential therapeutic agents in bone metastasis in patients with breast, prostate, and myeloma tumors, and clinical trials have shown efficacy and minimal systemic toxicity (18). Among the most potent bisphos- phonate compounds are nitrogen-containing bisphosphonates Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Yves A. DeClerck, Division of Hematology-Oncology, Children’s Hospital Los Angeles, MS#54, 4650 Sunset Boulevard, Los Angeles, CA 90027. Phone: 323-669-2150; Fax: 323-664-9455; E-mail: declerck@usc.edu. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-4508 Cancer Res 2007; 67: (19). October 1, 2007 9346 www.aacrjournals.org Research Article