Osteosarcoma (OS) is the most common primary malignancy of bone. Mortality is determined by the presence of metastatic disease, but little is known regarding the biochemical events that drive metastases. Two murine OS cell lines, K7M2 and K12, are related but differ significantly in their metastatic potentials: K7M2 is highly metastatic whereas K12 displays much less metastatic potential. Using this experimental system, the mammalian target of rapamycin (mTOR) pathway has been implicated in OS metastasis. We also discovered that aldehyde dehydrogenase (ALDH, a stem cell marker) activity is higher in K7M2 cells than K12 cells. Rapamycin treatment reduces the expression and enzymatic activity of ALDH in K7M2 cells. ALDH inhibition renders these cells more susceptible to apoptotic death when exposed to oxidative stress. Furthermore, rapamycin treatment reduces bone morphogenetic protein-2 (BMP2) and vascular endothelial growth factor (VEGF) gene expression and inhibits K7M2 proliferation, migration, and invasion in vitro. Inhibition of ALDH with disulfiram correlated with decreased mTOR expression and activity. In conclusion, we provide evidence for interaction between mTOR activity, ALDH activity, and metastatic potential in murine OS cells. Our work suggests that mTOR and ALDH are therapeutic targets for the treatment and prevention of OS metastasis. 1. Introduction Osteosarcoma (OS), the most common primary malignancy of bone, usually occurs in the long bones during childhood and adolescence at sites of rapid bone turnover [1–3]. Despite pre- and postoperative chemotherapy and wide surgical resection of the tumor, overall survival for patients without radiographically detectable metastases is only 65–70% [1, 2, 4, 5, 8]. The prognosis for patients with detectable metastases at the time of diagnosis is particularly poor, ranging from 15 to 30% [1, 7, 8]. It is thus the presence of pulmonary metastatic disease that ultimately determines OS mortality [9]. However, little is known about the biochemical signaling pathways that drive the progression of metastases and the molecular biology of OS remains poorly understood. As a result, we have yet to develop therapeutic strategies that specifically target metastatic disease. The mammalian target of rapamycin (mTOR) is a highly conserved serine/threonine kinase, and as its name implies, mTOR activity is specifically inhibited by the drug rapamycin [10–13]. Rapamycin is an antimicrobial agent produced by Streptomyces hygroscopius that also exhibits potent immunosuppressive and antitumor properties, likely due to
References
[1]
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.
[2]
D. Carrle and S. S. Bielack, “Current strategies of chemotherapy in osteosarcoma,” International Orthopaedics, vol. 30, no. 6, pp. 445–451, 2006.
[3]
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.
[4]
S. Ferrari, S. Smeland, M. Mercuri et al., “Neoadjuvant chemotherapy with high-dose ifosfamide, high-dose methotrexate, cisplatin, and doxorubicin for patients with localized osteosarcoma of the extremity: a joint study by the italian and Scandinavian Sarcoma Groups,” Journal of Clinical Oncology, vol. 23, no. 34, pp. 8845–8852, 2005.
[5]
P. J. Messerschmitt, R. M. Garcia, F. W. Abdul-Karim, E. M. Greenfield, and P. J. Getty, “Osteosarcoma,” Journal of the American Academy of Orthopaedic Surgeons, vol. 17, no. 8, pp. 515–527, 2009.
[6]
K. Vo, B. Amarasinghe, K. Washington, A. Gonzalez, J. Berlin, and T. P. Dang, “Targeting notch pathway enhances rapamycin antitumor activity in pancreas cancers through PTEN phosphorylation,” Molecular Cancer, vol. 10, article 138, 2011.
[7]
P. A. Meyers, C. L. Schwartz, M. Krailo et al., “Osteosarcoma: a randomized, prospective trial of the addition of ifosfamide and/or muramyl tripeptide to cisplatin, doxorubicin, and high-dose methotrexate,” Journal of Clinical Oncology, vol. 23, no. 9, pp. 2004–2011, 2005.
[8]
P. A. Meyers, C. L. Schwartz, M. D. Krailo et al., “Osteosarcoma: the addition of muramyl tripeptide to chemotherapy improves overall survival—a report from the children's oncology group,” Journal of Clinical Oncology, vol. 26, no. 4, pp. 633–638, 2008.
[9]
P. A. Meyers and R. Gorlick, “Osteosarcoma,” Pediatric Clinics of North America, vol. 44, no. 4, pp. 973–989, 1997.
[10]
E. J. Brown, M. W. Albers, T. B. Shin et al., “A mammalian protein targeted by G1-arresting rapamycin-receptor complex,” Nature, vol. 369, no. 6483, pp. 756–758, 1994.
[11]
M. I. Chiu, H. Katz, and V. Berlin, “RAPT1, a mammalian homolog of yeast Tor, interacts with the FKBP12/rapamycin complex,” Proceedings of the National Academy of Sciences of the United States of America, vol. 91, no. 26, pp. 12574–12578, 1994.
[12]
D. M. Sabatini, H. Erdjument-Bromage, M. Lui, P. Tempst, and S. H. Snyder, “RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs,” Cell, vol. 78, no. 1, pp. 35–43, 1994.
[13]
C. J. Sabers, M. M. Martin, G. J. Brunn et al., “Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells,” The Journal of Biological Chemistry, vol. 270, no. 2, pp. 815–822, 1995.
[14]
H. Zhou and S. Huang, “MTOR signaling in cancer cell motility and tumor metastasis,” Critical Reviews in Eukaryotic Gene Expression, vol. 20, no. 1, pp. 1–16, 2010.
[15]
S. Wullschleger, R. Loewith, and M. N. Hall, “TOR signaling in growth and metabolism,” Cell, vol. 124, no. 3, pp. 471–484, 2006.
[16]
R. Zoncu, A. Efeyan, and D. M. Sabatini, “MTOR: from growth signal integration to cancer, diabetes and ageing,” Nature Reviews Molecular Cell Biology, vol. 12, no. 1, pp. 21–35, 2011.
[17]
E. Dazert and M. N. Hall, “mTOR signaling in disease,” Current Opinion in Cell Biology, vol. 23, pp. 744–755, 2011.
[18]
E. Jacinto, R. Loewith, A. Schmidt et al., “Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive,” Nature Cell Biology, vol. 6, no. 11, pp. 1122–1128, 2004.
[19]
R. Loewith, E. Jacinto, S. Wullschleger et al., “Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control,” Molecular Cell, vol. 10, no. 3, pp. 457–468, 2002.
[20]
M. F. Crouch, “Regulation of thrombin-induced stress fibre formation in Swiss 3T3 cells by the 70-kDa S6 kinase,” Biochemical and Biophysical Research Communications, vol. 233, no. 1, pp. 193–199, 1997.
[21]
A. Dufner and G. Thomas, “Ribosomal S6 kinase signaling and the control of translation,” Experimental Cell Research, vol. 253, no. 1, pp. 100–109, 1999.
[22]
S. Ferrari, H. R. Bandi, J. Hofsteenge, B. M. Bussian, and G. Thomas, “Mitogen-activated 70K S6 kinase. Identification of in vitro 40 S ribosomal S6 phosphorylation sites,” The Journal of Biological Chemistry, vol. 266, no. 33, pp. 22770–22775, 1991.
[23]
Q. Zhou, Z. Deng, Y. Zhu, H. Long, S. Zhang, and J. Zhao, “mTOR/p70S6K signal transduction pathway contributes to osteosarcoma progression and patients' prognosis,” Medical Oncology, vol. 27, no. 4, pp. 1239–1245, 2010.
[24]
C. Khanna, J. Prehn, C. Yeung, J. Caylor, M. Tsokos, and L. Helman, “An orthotopic model of murine osteosarcoma with clonally related variants differing in pulmonary metastatic potential,” Clinical and Experimental Metastasis, vol. 18, no. 3, pp. 261–271, 2000.
[25]
X. Wan, A. Mendoza, C. Khanna, and L. J. Helman, “Rapamycin inhibits ezrin-mediated metastatic behavior in a murine model of osteosarcoma,” Cancer Research, vol. 65, no. 6, pp. 2406–2411, 2005.
[26]
E. Charafe-Jauffret, C. Ginestier, F. Iovino et al., “Aldehyde dehydrogenase 1-positive cancer stem cells mediate metastasis and poor clinical outcome in inflammatory breast cancer,” Clinical Cancer Research, vol. 16, no. 1, pp. 45–55, 2010.
[27]
A. M. S. Cheung, T. S. K. Wan, J. C. K. Leung et al., “Aldehyde dehydrogenase activity in leukemic blasts defines a subgroup of acute myeloid leukemia with adverse prognosis and superior NOD/SCID engrafting potential,” Leukemia, vol. 21, no. 7, pp. 1423–1430, 2007.
[28]
H. J. Cho, T. S. Lee, J. B. Park et al., “Disulfiram suppresses invasive ability of osteosarcoma cells via the inhibition of MMP-2 and MMP-9 expression,” Journal of Biochemistry and Molecular Biology, vol. 40, no. 6, pp. 1069–1076, 2007.
[29]
K. Honoki, H. Fujii, A. Kubo et al., “Possible involvement of stem-like populations with elevated ALDH1 in sarcomas for chemotherapeutic drug resistance,” Oncology Reports, vol. 24, no. 2, pp. 501–505, 2010.
[30]
E. H. Huang, M. J. Hynes, T. Zhang et al., “Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis,” Cancer Research, vol. 69, no. 8, pp. 3382–3389, 2009.
[31]
K. R. Weiss, G. M. Cooper, J. A. Jadlowiec, R. L. McGough III, and J. Huard, “VEGF and BMP expression in mouse osteosarcoma cells,” Clinical Orthopaedics and Related Research, vol. 450, pp. 111–117, 2006.
[32]
M. Magni, S. Shammah, R. Schiró, W. Mellado, R. Dalla-Favera, and A. M. Gianni, “Induction of cyclophosphamide-resistance by aldehyde-dehydrogenase gene transfer,” Blood, vol. 87, no. 3, pp. 1097–1103, 1996.
[33]
J. E. Russo and J. Hilton, “Characterization of cytosolic aldehyde dehydrogenase from cyclophosphamide resistant L1210 cells,” Cancer Research, vol. 48, no. 11, pp. 2963–2968, 1988.
[34]
M. W. Albers, R. T. Williams, E. J. Brown, A. Tanaka, F. L. Hall, and S. L. Schreiber, “FKBP-rapamycin inhibits a cyclin-dependent kinase activity and a cyclin D1-Cdk association in early G1 of an osteosarcoma cell line,” The Journal of Biological Chemistry, vol. 268, no. 30, pp. 22825–22829, 1993.
[35]
L. M. Machesky, “Lamellipodia and filopodia in metastasis and invasion,” FEBS Letters, vol. 582, no. 14, pp. 2102–2111, 2008.
[36]
C. Khanna, J. Khan, P. Nguyen et al., “Metastasis-associated differences in gene expression in a murine model of osteosarcoma,” Cancer Research, vol. 61, no. 9, pp. 3750–3759, 2001.
[37]
L. Ren, S. H. Hong, Q. R. Chen et al., “Dysregulation of ezrin phosphorylation prevents metastasis and alters cellular metabolism in osteosarcoma,” Cancer Research, vol. 72, pp. 1001–1012, 2012.
[38]
M. S. Wicha, S. Liu, and G. Dontu, “Cancer stem cells: an old idea—a paradigm shift,” Cancer Research, vol. 66, no. 4, pp. 1883–1890, 2006.
[39]
Y. Y. Hu, M. H. Zheng, R. Zhang, Y. M. Liang, and H. Han, “Notch signaling pathway and cancer metastasis,” Advances in Experimental Medicine and Biology, vol. 727, pp. 186–198, 2012.
[40]
J. Zhou, P. Su, L. Wang et al., “mTOR supports long-term self-renewal and suppresses mesoderm and endoderm activities of human embryonic stem cells,” Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 19, pp. 7840–7845, 2009.
[41]
P. Sampath, D. K. Pritchard, L. Pabon et al., “A hierarchical network controls protein translation during murine rmbryonic stem cell self-renewal and differentiation,” Cell Stem Cell, vol. 2, no. 5, pp. 448–460, 2008.
[42]
Y. Gazitt, V. Kolaparthi, K. Moncada, C. Thomas, and J. Freeman, “Targeted therapy of human osteosarcoma with 17AAG or rapamycin: characterization of induced apoptosis and inhibition of mTOR and Akt/MAPK/Wnt pathways,” International Journal of Oncology, vol. 34, no. 2, pp. 551–561, 2009.
[43]
T. Ogawa, M. Tokuda, K. Tomizawa et al., “Osteoblastic differentiation is enhanced by rapamycin in rat osteoblast-like osteosarcoma (ROS 17/2.8) cells,” Biochemical and Biophysical Research Communications, vol. 249, no. 1, pp. 226–230, 1998.
[44]
C. P. Gibbs Jr., P. P. Levings, and S. C. Ghivizzani, “Evidence for the osteosarcoma stem cell,” Current Orthopaedic Practice, vol. 22, pp. 322–326, 2011.
[45]
W. P. Tseng, S. N. Yang, C. H. Lai, and C. H. Tang, “Hypoxia induces BMP-2 expression via ILK, Akt, mTOR, and HIF-1 pathways in osteoblasts,” Journal of Cellular Physiology, vol. 223, no. 3, pp. 810–818, 2010.
[46]
L. M. Ellis and D. J. Hicklin, “VEGF-targeted therapy: mechanisms of anti-tumour activity,” Nature Reviews Cancer, vol. 8, no. 8, pp. 579–591, 2008.
[47]
M. Guba, P. Von Breitenbuch, M. Steinbauer et al., “Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor,” Nature Medicine, vol. 8, no. 2, pp. 128–135, 2002.
