Osteosarcoma (OS) is the most common primary bone malignancy with a high propensity for local invasion and distant metastasis. Despite current multidisciplinary treatments, there has not been a drastic change in overall prognosis within the past 2 decades. Dickkopf-3 protein (Dkk-3/REIC) has been known to inhibit canonical Wnt/β-catenin pathway, and its expression has been shown to be downregulated in OS cell lines. Using in vivo and in vitro studies, we demonstrated that Dkk-3-transfected 143B cells inhibited tumorigenesis and metastasis in an orthotopic xenograft model of OS. Inoculation of Dkk-3-transfected 143B cell lines into nude mice showed significant decreased tumor growth and less metastatic pulmonary nodules (88.7%) compared to the control vector. In vitro experiments examining cellular motility and viability demonstrated less anchorage-independent growth and decreased cellular motility for Dkk-3-transfected 143B and SaOS2 cell lines compared to the control vector. Downstream expressions of Met, MAPK, ALK, and S1004A were also downregulated in Dkk-3-transfected SaOS2 cells, suggesting the ability of Dkk-3 to inhibit tumorigenic potential of OS. Together, these data suggest that Dkk-3 has a negative impact on the progression of osteosarcoma. Reexpressing Dkk-3 in Dkk-3-deficient OS tumors may prove to be of benefit as a preventive or therapeutic strategy. 1. Introduction Osteosarcoma (OS) is the most common primary bone malignancy diagnosed in children and adolescents. With the current multidisciplinary treatments, 60–70% of patients with localized disease survive [1]. According to the Children’s Oncology Group (COG) protocol for localized disease, standard therapy consists of neoadjuvant chemotherapy, including doxorubicin, cisplatin, and high-dose methotrexate, followed by surgical resection. After surgical intervention, adjuvant chemotherapy is given, dependent upon the degree of necrosis. Good responders to neoadjuvant therapy will show < 10% viable tumor and will be randomized to continue with adjuvant therapy. According to the European and American Osteosarcoma 1 Trial (EURAMOS 1), the five-year survival for good responders is 75–80% compared to poor responders who face survival percentages of 45–55%. Prognostic factors for OS include tumor site and size, primary metastases, response to chemotherapy and surgical remission [2, 3]. Osteosarcoma has a high tendency for local invasion and early metastasis. Unfortunately, with metastatic disease, the rate of 5-year overall survival is greatly reduced to 20–30%, and the 5-year event-free
References
[1]
G. Bacci, S. Ferrari, F. Bertoni et al., “Long-term outcome for patients with nonmetastatic osteosarcoma of the extremity treated at the istituto ortopedico rizzoli according to the istituto ortopedico rizzoli/osteosarcoma-2 protocol: an updated report,” Journal of Clinical Oncology, vol. 18, no. 24, pp. 4016–4027, 2000.
[2]
S. S. Bielack, B. Kempf-Bielack, G. Delling et al., “Prognostic factors in high-grade osteosarcoma of the extremities or trunk: an analysis of 1,702 patients treated on neoadjuvant cooperative osteosarcoma study group protocols,” Journal of Clinical Oncology, vol. 20, no. 3, pp. 776–790, 2002.
[3]
L. Kager, A. Zoubek, U. P？tschger et al., “Primary metastatic osteosarcoma: presentation and outcome of patients treated on neoadjuvant cooperative osteosarcoma study group protocols,” Journal of Clinical Oncology, vol. 21, no. 10, pp. 2011–2018, 2003.
[4]
V. Mialou, T. Philip, C. Kalifa et al., “Metastatic osteosarcoma at diagnosis: prognostic factors and long-term outcome—the French pediatric experience,” Cancer, vol. 104, no. 5, pp. 1100–1109, 2005.
[5]
J. B. Hayden and B. H. Hoang, “Osteosarcoma: basic science and clinical implications,” Orthopedic Clinics of North America, vol. 37, no. 1, pp. 1–7, 2006.
[6]
B. T. MacDonald, K. Tamai, and X. He, “Wnt/β-catenin signaling: components, mechanisms, and diseases,” Developmental Cell, vol. 17, no. 1, pp. 9–26, 2009.
[7]
R. T. Moon, “Wnt/beta-catenin pathway,” Science's STKE, vol. 2005, no. 271, article cm1, 2005.
[8]
C. Jamieson, M. Sharma, and B. R. Henderson, “Wnt signaling from membrane to nucleus: beta-catenin caught in a loop,” The International Journal of Biochemistry & Cell Biology, vol. 44, no. 6, pp. 847–850, 2012.
[9]
H. H. Luu, R. Zhang, R. C. Haydon et al., “Wnt/β-catenin signaling pathway as novel cancer drug targets,” Current Cancer Drug Targets, vol. 4, no. 8, pp. 653–671, 2004.
[10]
B. H. Hoang, T. Kubo, J. H. Healey et al., “Dickkopf 3 inhibits invasion and motility of saos-2 osteosarcoma cells by modulating the Wnt-β-catenin pathway,” Cancer Research, vol. 64, no. 8, pp. 2734–2739, 2004.
[11]
S. Y. Hsieh, P. S. Hsieh, C. T. Chiu, and W. Y. Chen, “Dickkopf-3/REIC functions as a suppressor gene of tumor growth,” Oncogene, vol. 23, no. 57, pp. 9183–9189, 2004.
[12]
K. Zhang, M. Watanabe, Y. Kashiwakura et al., “Expression pattern of REIC/Dkk-3 in various cell types and the implications of the soluble form in prostatic acinar development,” International Journal of Oncology, vol. 37, no. 6, pp. 1495–1501, 2010.
[13]
I. L. Jung, J. K. Hyo, C. K. Kug, and G. K. In, “Knockdown of the Dickkopf 3 gene induces apoptosis in a lung adenocarcinoma,” International Journal of Molecular Medicine, vol. 26, no. 1, pp. 33–38, 2010.
[14]
S. Kuphal, S. Lodermeyer, F. Bataille, M. Schuierer, B. H. Hoang, and A. K. Bosserhoff, “Expression of Dickkopf genes is strongly reduced in malignant melanoma,” Oncogene, vol. 25, no. 36, pp. 5027–5036, 2006.
[15]
P. Polakis, “Wnt signaling and cancer,” Genes and Development, vol. 14, no. 15, pp. 1837–1851, 2000.
[16]
Y. Mizobuchi, K. Matsuzaki, K. Kuwayama et al., “REIC/Dkk-3 induces cell death in human malignant glioma,” Neuro-Oncology, vol. 10, no. 3, pp. 244–253, 2008.
[17]
K. Ueno, H. Hirata, S. Majid et al., “Wnt antagonist DICKKOPF-3 (Dkk-3) induces apoptosis in human renal cell carcinoma,” Molecular Carcinogenesis, vol. 50, no. 6, pp. 449–457, 2011.
[18]
C. Zenzmaier, G. Untergasser, M. Hermann, S. Dirnhofer, N. Sampson, and P. Berger, “Dysregulation of Dkk-3 expression in benign and malignant prostatic tissue,” Prostate, vol. 68, no. 5, pp. 540–547, 2008.
[19]
R. Tanimoto, F. Abarzua, M. Sakaguchi et al., “REIC/Dkk-3 as a potential gene therapeutic agent against human testicular cancer,” International Journal of Molecular Medicine, vol. 19, no. 3, pp. 363–368, 2007.
[20]
Z. R. Yang, W. G. Dong, X. F. Lei, et al., “Overexpression of Dickkopf-3 induces apoptosis through mitochondrial pathway in human colon cancer,” World Journal of Gastroenterology, vol. 18, no. 14, pp. 1590–1601, 2012.
[21]
Y. Guo, X. Zi, Z. Koontz et al., “Blocking Wnt/LRP5 signaling by a soluble receptor modulates the epithelial to mesenchymal transition and suppresses met and metalloproteinases in osteosarcoma Saos-2 cells,” Journal of Orthopaedic Research, vol. 25, no. 7, pp. 964–971, 2007.
[22]
J. Veeck and E. Dahl, “Targeting the Wnt pathway in cancer: the emerging role of Dickkopf-3,” Biochimica et Biophysica Acta, vol. 1825, no. 1, pp. 18–28, 2012.
[23]
B. Wu, S. P. Crampton, and C. C. W. Hughes, “Wnt Signaling induces matrix metalloproteinase expression and regulates T cell transmigration,” Immunity, vol. 26, no. 2, pp. 227–239, 2007.
[24]
M. Uchibori, Y. Nishida, T. Nagasaka, Y. Yamada, K. Nakanishi, and N. Ishiguro, “Increased expression of membrane-type matrix metalloproteinase-1 is correlated with poor prognosis in patients with osteosarcoma,” International Journal of Oncology, vol. 28, no. 1, pp. 33–42, 2006.
[25]
K. F. Becker, E. Rosivatz, K. Blechschmidt, E. Kremmer, M. Sarbia, and H. H？fler, “Analysis of the E-cadherin repressor snail in primary human cancers,” Cells Tissues Organs, vol. 185, no. 1–3, pp. 204–212, 2007.
[26]
P. McQueen, S. Ghaffar, Y. Guo, et al., “The Wnt signaling pathway: implications for therapy in osteosarcoma,” Expert Review of Anticancer Therapy, vol. 11, no. 8, pp. 1223–1232, 2011.
[27]
Y. Kang and J. Massagué, “Epithelial-mesenchymal transitions: twist in development and metastasis,” Cell, vol. 118, no. 3, pp. 277–279, 2004.
[28]
L. R. Howe, O. Watanabe, J. Leonard, and A. M. C. Brown, “Twist is up-regulated in response to Wnt1 and inhibits mouse mammary cell differentiation,” Cancer Research, vol. 63, no. 8, pp. 1906–1913, 2003.
[29]
S. C. Garrett, K. M. Varney, D. J. Weber, and A. R. Bresnick, “S100A4, a mediator of metastasis,” Journal of Biological Chemistry, vol. 281, no. 2, pp. 677–680, 2006.
[30]
M. Fujiwara, T. G. Kashima, A. Kunita et al., “Stable knockdown of S100A4 suppresses cell migration and metastasis of osteosarcoma,” Tumour Biology, vol. 32, no. 3, pp. 611–622, 2011.
[31]
G. Zhang, M. Li, J. Jin, et al., “Knockdown of S100A4 decreases tumorigenesis and metastasis in osteosarcoma cells by repression of matrix metalloproteinase-9,” Asian Pacific Journal of Cancer Prevention, vol. 12, no. 8, pp. 2075–2080, 2011.
[32]
N. Coltella, M. C. Manara, V. Cerisano et al., “Role of the MET/HGF receptor in proliferation and invasive behavior of osteosarcoma,” The FASEB Journal, vol. 17, no. 9, pp. 1162–1164, 2003.
[33]
B. H. Hoang, T. Kubo, J. H. Healey et al., “Expression of LDL receptor-related protein 5 (LRP5) as a novel marker for disease progression in high-grade osteosarcoma,” International Journal of Cancer, vol. 109, no. 1, pp. 106–111, 2004.
