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Case Orthop J. Author manuscript; available in PMC 2015 December 28. Published in final edited form as: Case Orthop J. 2013 ; 10(1): 38–42.
CANCER STEM CELLS IN OSTEOSARCOMA Lindsay Bashur, PhD and Guang Zhou, PhD Department of Orthopaedic Surgery, Case Western Reserve University
Abstract
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Osteosarcoma is the most common type of bone cancer and the second leading cause of cancerrelated deaths in pediatric patients. Despite conventional treatments such as surgery and chemotherapy, long-term survival rates for patients diagnosed with osteosarcoma have not improved over the last 30 years, likely due to drug-resistant metastasis and disease recurrence. An emerging concept in cancer research is that within a heterogeneous tumor there is a small subset of cells called “cancer stem cells” that are responsible for drug resistance, tumor recurrence and metastasis. This brief review summarizes our current knowledge about cancer stem cells in osteosarcoma, including their potential as a new target for osteosarcoma treatment.
Osteosarcoma
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Osteosarcoma, the most common primary bone sarcoma, mainly occurs in children and adolescents.1,2 Osteosarcomas usually develop in areas of rapid bone growth or turnover in the long bones, such as the distal femur, proximal tibia, and proximal humerus.3 In the United States, approximately 600 cases of osteosarcoma are diagnosed per year, making osteosarcoma the fifth most frequent malignancy in 15- to 19-year-olds.3-5 Osteosarcoma can also occur in adults over 65 years of age who have rapid bone turnover due to conditions like Paget's disease.6 Patients with localized osteosarcoma have a 5-year survival rate of 70% with chemotherapy in combination with surgery.3,7 Approximately 30-40% of osteosarcoma patients, however, eventually develop metastases, most commonly in the lungs.7,8 The long-term survival rate of osteosarcoma patients with metastases is only 20-30% and has not improved much over the past 30 years.9 A better understanding of the origin and progression of osteosarcoma is essential for the development of new diagnostic and treatment strategies to increase the long-term survival rate.
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The exact causes of osteosarcoma are not completely understood. Growing evidence, however, suggests that osteosarcoma arises from mesenchymal cells, which, like osteoblasts, have the ability to produce osteoid.6 Osteoblast differentiation from mesenchymal cells is a tightly regulated process that is critical for proper bone formation (Figure 1A). Several transcription factors, such as Runx2, Osterix, and Sox9, are essential for proper osteoblast differentiation. Consequently, if the transcriptional network controlling the lineage differentiation of mesenchymal cells into osteoblasts is disrupted, then tumorigenic precursor cells are likely formed, potentially leading to the development of osteosarcoma (Figure 1B).10 Furthermore, inactivating mutations of tumor suppressor genes p53 and pRb are also intimately involved in osteosarcoma development. p53 is a well-known tumor suppressor
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that is mutated in more than half of primary human tumors. p53 also plays an important role in osteoblast differentiation. Indeed, p53−/− mice exhibited increased bone mass and bone formation, and p53−/− osteoblasts showed accelerated differentiation and up-regulated expression of osteogenic transcription factor Osterix.11 Moreover, the inhibition of p53 function mediated by oncogenic factor Mdm2 is a prerequisite for Runx2 activation and proper skeletal formation.12 In recent years, the Osterix-Cre transgenic mouse model, which targets osteoblast precursor cells during development, has been used to generate novel mouse models for osteosarcoma. When both p53 and pRb were deleted using the Osx-Cre mice, osteosarcoma occurred with almost 100% penetrance.13,14 Further analysis revealed that osteosarcoma development is likely to be initiated by the loss of p53 and potentiated by the loss of pRb.13,14
Cancer Stem Cell Hypothesis Author Manuscript
Heterogeneity is a defining feature of solid tumors. Several factors contribute to this heterogeneity, including genetic mutations, epigenetic changes, interactions with the microenvironment, cellular morphology, and proliferation and differentiation capacity.15,16 The cancer stem cell (CSC) hypothesis proposes that a heterogeneous tumor contains a hierarchal organization of cells in which only a small subset called CSCs are responsible for sustaining tumor growth.17 The first evidence of CSCs came from studies in acute myeloid leukemia (AML). In 1994, Lapidot et al. demonstrated that only a small percentage of AML cells were capable of generating leukemia when transplanted into immunodeficient mice.18 Since then, CSCs or CSC-like cells have been identified in several types of cancers, including breast19, brain20, colon21, prostate22, and bone23.
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CSCs are generally defined as tumor cells with stem-like characteristics that are responsible for sustaining tumor growth. Similar to normal stem cells, CSCs have self-renewal potential and differentiation capacity.17,24 The CSC division process is also similar to that of normal stem cells. CSCs can divide through either symmetric or asymmetric division, although primarily asymmetric division. During asymmetric division, CSCs divide to produce two daughter cells: an identical CSC with self-renewal capacity and a differentiated progenitor cell responsible for the majority of cell division (Figure 2).25 The exact origin of CSCs is uncertain. CSCs may arise from either normal stem cells or more differentiated cells that have acquired stem cell-like properties during malignant transformation.26
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In addition, evidence suggests that CSCs are resistant to cancer therapies such as chemotherapy and radiation. CSCs have several properties that make them resistant to traditional cancer therapies, including enhanced DNA repair ability, quiescence, increased expression of drug efflux transporters, and increased resistance to apoptosis.25 Traditional cancer therapies, therefore, may reduce the majority of the primary tumor but actually miss the small subset of CSCs. It is possible that CSCs are drug-resistant and consequently responsible for tumor recurrence and metastasis formation. Thus, the development of CSCtargeted therapies is important to prevent primary tumor recurrence and metastasis.
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Cancer Stem Cells in Osteosarcoma
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Recent studies have identified the existence of CSC-like cells in osteosarcoma. Gibbs et al. were the first to isolate a CSC subpopulation from nine biopsies of untreated osteosarcoma and the MG-63 osteosarcoma cell line using a sphere formation assay.23 The cells grew in spherical colonies, also called sarcospheres, when cultured with epidermal growth factor and basic fibroblast growth factor under serum starvation and anchorage-independent conditions. The sarcosphere-derived cells had increased expression of the stem cell markers Oct4 and Nanog compared with adherent cells. These sarcospheres were also shown to have the ability to self-renew through the formation of secondary spheres. Several other groups have since confirmed the capability of isolating CSCs expressing Oct4 and Nanog from osteosarcoma using sphere culture.27,28 In addition to Oct4 and Nanog, the stem cell transcription factor Sox2 has also been shown to be required for osteosarcoma selfrenewal.29 Using the MG-63 osteosarcoma cell line model, it has been shown that MG-63 spheres also have a higher resistance to chemotherapy drugs and a greater expression of DNA mismatch repair enzymes than adherent MG-63 cells.27 To date, sphere culture is the most widely used method for isolating osteosarcoma CSC-like cells. Sphere formation, however, can also occur within non-stem cell populations.30 Therefore, a more detailed analysis is needed to better characterize and identify osteosarcoma CSCs.
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CSC-like cells from osteosarcoma have also been isolated by fluorescent and magnetic cell sorting for CD133+ (prominin), CD117+ (c-kit), and Stro-1+ 2. Tirino et al. identified a small population of CD133+ cells in several human osteosarcoma cell lines with stem-like characteristics including a high proliferation rate and the ability to form spheres in culture.31 CD117+ Stro-1+ cells have been shown to have increased tumorigenicity when injected subcutaneously into mice, increased metastasis after orthotopic injections, and drug-resistant properties.32 Recently, Ying et al. used an inverse-lineage tracking strategy coupled with serial human-to-mouse xenotransplantation to identify osteosarcoma cells with CSC-like properties. They found that CD49f− CD133+ cells had CSC-like properties including selfrenewal and strong tumorigenicity that correlated with diminished osteogenic fate, while CD49f+ had limited tumorigenicity and more differentiated osteogenic features.33 High aldehyde dehydrogenase (ALDH) activity has also been used to identify CSC-like cells in osteosarcoma.2
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In addition to osteosarcoma, CSC-like cells have also been characterized in other sarcomas, including Ewing sarcoma and rhabdomyosarcoma, using sphere formation and marker analysis of CD133 and ALDH.26 Ewing sarcoma is the second most common malignant bone tumor and, similar to osteosarcoma, is associated with poor prognosis and low survival rates. Suva et al. isolated a subpopulation of CD133+ tumor cells from primary Ewing sarcoma tumors.34 They showed that these cells were able to initiate and sustain tumor growth through serial transplantation in immunodeficient mice. The CD133+ cells also expressed higher levels of stem cell markers Oct4 and Nanog than the CD133− cells. One major challenge in the osteosarcoma CSC field is to identify reliable and consensus CSC markers.2,26,35 One reason for the variability among different studies is that the markers expressed by CSC may differ among patients.36 Thus, putative osteosarcoma CSC
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markers need to be tested in a significant number of patients to determine how to assign those markers based on the type, subtype, or stage of cancer, as well as how to distinguish tumorigenic cells from nontumorigenic cells in different tumors. It will also be important to perform lineage tracing studies in order to determine the origin and characteristics of the CSCs that contribute to tumor growth and disease progression.
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Yang et al. recently reported what may be the first CSCs discovered in bone in a mouse model.37 Metachondromatosis is a benign cartilage-tumor syndrome caused by uncontrolled chondrocyte proliferation in humans, resulting from loss-of-function mutations in PTPN11. The authors deleted Ptpn11 in mice and, using lineage tracing studies, identified a population of progenitor cells in the perichondrium tissue layer that surrounds cartilage in long bones.37 These cells, similar to mesenchymal stem cells, can differentiate into several cell types including osteoblasts, chondrocytes, and adipocytes, in vitro. They have shown that Ptpn11 acts as a tumor suppressor in these CSC-like cells; therefore, the deletion of Ptpn11 initiates the formation of cartilage tumors.
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The transcriptional cofactor JAB1 is highly expressed in many different types of cancers, and has emerged as a novel player in tumorigenesis through its effects on cell proliferation, differentiation, survival, and cell cycle progression.38 Our laboratory is studying the potential role of JAB1 in the pathogenesis of osteosarcoma. We have confirmed that JAB1 is highly expressed in biopsy samples of osteosarcoma patients. In addition, JAB1 knockdown in the metastatic 143B osteosarcoma cell-line exhibits significantly reduced cell proliferation, colony formation, and motility, suggesting that down-regulating JAB1 expression in osteosarcoma reduces tumorigenesis. The underlying mechanisms of JAB1 involvement in tumor development, however, remain poorly understood. We have previously shown that Jab1 plays critical roles in the successive steps of skeletogenesis in vivo.39 Further elucidation of the crucial roles of Jab1 in osteoprogenitor cells during skeletal development may provide insights into the effect of JAB1 on key CSC properties, such as stem cell maintenance and renewal, and may eventually lead to the development of JAB1-targeted cancer therapies.
Conclusion
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The poor prognosis of osteosarcoma patients is related to drug resistance, and osteosarcoma cancer stem cells are proposed to be at least partly responsible for this drug resistance. The development of CSC-targeted therapy, therefore, can potentially prevent tumor recurrence and increase survival rates of osteosarcoma patients. However, CSC research is still in its infancy, especially for osteosarcoma and other bone cancers, and many challenges still remain. Future research is needed to determine an optimal method for osteosarcoma stem cell identification and isolation, and to further understand the mechanisms of CSC selfrenewal, metastasis, and drug resistance.
Acknowledgements We thank Valerie Schmedlen for editorial assistance.
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References
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21. Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, De Maria R. Identification and expansion of human colon-cancer-initiating cells. Nature. 2007; 445(7123):111– 5. [PubMed: 17122771] 22. Collins AT, Berry PA, Hyde C, Stower MJ, Maitland NJ. Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res. 2005; 65(23):10946–51. [PubMed: 16322242] 23. Gibbs CP, Kukekov VG, Reith JD, Tchigrinova O, Suslov ON, Scott EW, Ghivizzani SC, Ignatova TN, Steindler DA. Stem-like cells in bone sarcomas: implications for tumorigenesis. Neoplasia. 2005; 7(11):967–76. [PubMed: 16331882] 24. Baccelli I, Trumpp A. The evolving concept of cancer and metastasis stem cells. J Cell Biol. 2012; 198(3):281–93. [PubMed: 22869594] 25. Liu B, Ma W, Jha RK, Gurung K. Cancer stem cells in osteosarcoma: recent progress and perspective. Acta Oncol. 2011; 50(8):1142–50. [PubMed: 21718210] 26. Dela Cruz FS. Cancer stem cells in pediatric sarcomas. Front Oncol. 2013; 3:168. [PubMed: 23819111] 27. Fujii H, Honoki K, Tsujiuchi T, Kido A, Yoshitani K, Takakura Y. Sphere-forming stem-like cell populations with drug resistance in human sarcoma cell lines. Int J Oncol. 2009; 34(5):1381–6. [PubMed: 19360350] 28. Wang L, Park P, Lin CY. Characterization of stem cell attributes in human osteosarcoma cell lines. Cancer Biol Ther. 2009; 8(6):543–52. [PubMed: 19242128] 29. Basu-Roy U, Seo E, Ramanathapuram L, Rapp TB, Perry JA, Orkin SH, Mansukhani A, Basilico C. Sox2 maintains self renewal of tumor-initiating cells in osteosarcomas. Oncogene. 2012; 31(18):2270–82. [PubMed: 21927024] 30. Pastrana E, Silva-Vargas V, Doetsch F. Eyes wide open: a critical review of sphere-formation as an assay for stem cells. Cell Stem Cell. 2011; l8(5):486–98. [PubMed: 21549325] 31. Tirino V, Desiderio V, d'Aguino R, De Francesco F, Pirrozi G, Graziano A, Galderisi U, Cavaliere C, De Rosa A, Papaccio G, Giordano A. Detection and characterization of CD133+ cancer stem cells in human solid tumours. PLoS One. 2008; 3(10):e3469. [PubMed: 18941626] 32. Adhikari AS, Agarwal N, Wood BM, Porretta C, Ruiz B, Pochampally RR, Iwakuma T. CD117 and Stro-1 identify osteosarcoma tumor-initiating cells associated with metastasis and drug resistance. Cancer Res. 2010; 70(11):4602–12. [PubMed: 20460510] 33. Ying M, Liu G, Shimada H, Ding W, May WA, He Q, Adams GB, Wu L. Human osteosarcoma CD49f(-)CD133(+) cells: impaired in osteogenic fate while gain of tumorigenicity. Oncogene. 2013; 32(36):4252–63. [PubMed: 23045288] 34. Suvá ML, Riggi N, Stehle JL, Baumer K, Tercier S, Joseph JM, Suvá D, Clément V, Provero P, Cironi L, Osterheld MC, Guillou L, Stamenkovic I. Identification of cancer stem cells in Ewing's sarcoma. Cancer Res. 2009; 69(5):1776–81. [PubMed: 19208848] 35. Basu-Roy U, Basilico C, Mansukhani A. Perspectives on cancer stem cells in osteosarcoma. Cancer Lett. 2013; 338(1):158–67. [PubMed: 22659734] 36. Meacham CE, Morrison SJ. Tumour heterogeneity and cancer cell plasticity. Nature. 2013; 501(7467):328–37. [PubMed: 24048065] 37. Yang W, Wang J, Moore DC, Liang H, Dooner M, Wu Q, Terek R, Chen Q, Ehrlich MG, Quesenberry PJ, Neel BG. Ptpn11 deletion in a novel progenitor causes metachondromatosis by inducing hedgehog signalling. Nature. 2013; 499(7459):491–5. [PubMed: 23863940] 38. Shackleford TJ, Claret FX. JAB1/CSN5: a new player in cell cycle control and cancer. Cell Div. 2010; 5:26. [PubMed: 20955608] 39. Chen D, Bashur LA, Liang B, Panatonni M, Tamai K, Pardi R, Zhou G. The transcriptional coregulator Jab1 is crucial for chondrocyte differentiation in vivo. J Cell Sci. 2013; 126(Pt 1):234– 43. [PubMed: 23203803] 40. Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature. 2001; 414(6859):105–11. [PubMed: 11689955]
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Figure 1.
Disruption in osteoblast differentiation may lead to the development of osteosarcoma. (A) Progression of normal osteogenic differentiation. Mesenchymal stem cells (MSCs) are pluripotent cells that can differentiate into many cell types. During a tightly regulated process controlling the balance between cell proliferation and cell differentiation, MSCs can eventually differentiate into mature osteoblasts. (B) When osteogenic differentiation is disrupted, osteosarcoma precursors may form, which can lead to osteosarcoma. This disruption may be caused by genetic changes, such as, the inactivation of tumor suppressors p53 and Rb, and/or epigenetic changes. These changes can lead to increased proliferation and decreased differentiation of cancer cells. (adapted from [10])
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Figure 2.
Cancer stem cell (CSC) hypothesis model. CSCs divide into two cells: 1) an identical CSC which self-renews and maintains a pool of CSCs (white balls), and 2) a differentiated progenitor cell that continues to proliferate generating heterogeneous cancer cells comprising the bulk of the tumor (grey balls). (adapted from [40])
Author Manuscript Author Manuscript Case Orthop J. Author manuscript; available in PMC 2015 December 28.
Case Orthop J. Author manuscript; available in PMC 2015 December 28. Published in final edited form as: Case Orthop J. 2013 ; 10(1): 38–42.
CANCER STEM CELLS IN OSTEOSARCOMA Lindsay Bashur, PhD and Guang Zhou, PhD Department of Orthopaedic Surgery, Case Western Reserve University
Abstract
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Osteosarcoma is the most common type of bone cancer and the second leading cause of cancerrelated deaths in pediatric patients. Despite conventional treatments such as surgery and chemotherapy, long-term survival rates for patients diagnosed with osteosarcoma have not improved over the last 30 years, likely due to drug-resistant metastasis and disease recurrence. An emerging concept in cancer research is that within a heterogeneous tumor there is a small subset of cells called “cancer stem cells” that are responsible for drug resistance, tumor recurrence and metastasis. This brief review summarizes our current knowledge about cancer stem cells in osteosarcoma, including their potential as a new target for osteosarcoma treatment.
Osteosarcoma
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Osteosarcoma, the most common primary bone sarcoma, mainly occurs in children and adolescents.1,2 Osteosarcomas usually develop in areas of rapid bone growth or turnover in the long bones, such as the distal femur, proximal tibia, and proximal humerus.3 In the United States, approximately 600 cases of osteosarcoma are diagnosed per year, making osteosarcoma the fifth most frequent malignancy in 15- to 19-year-olds.3-5 Osteosarcoma can also occur in adults over 65 years of age who have rapid bone turnover due to conditions like Paget's disease.6 Patients with localized osteosarcoma have a 5-year survival rate of 70% with chemotherapy in combination with surgery.3,7 Approximately 30-40% of osteosarcoma patients, however, eventually develop metastases, most commonly in the lungs.7,8 The long-term survival rate of osteosarcoma patients with metastases is only 20-30% and has not improved much over the past 30 years.9 A better understanding of the origin and progression of osteosarcoma is essential for the development of new diagnostic and treatment strategies to increase the long-term survival rate.
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The exact causes of osteosarcoma are not completely understood. Growing evidence, however, suggests that osteosarcoma arises from mesenchymal cells, which, like osteoblasts, have the ability to produce osteoid.6 Osteoblast differentiation from mesenchymal cells is a tightly regulated process that is critical for proper bone formation (Figure 1A). Several transcription factors, such as Runx2, Osterix, and Sox9, are essential for proper osteoblast differentiation. Consequently, if the transcriptional network controlling the lineage differentiation of mesenchymal cells into osteoblasts is disrupted, then tumorigenic precursor cells are likely formed, potentially leading to the development of osteosarcoma (Figure 1B).10 Furthermore, inactivating mutations of tumor suppressor genes p53 and pRb are also intimately involved in osteosarcoma development. p53 is a well-known tumor suppressor
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that is mutated in more than half of primary human tumors. p53 also plays an important role in osteoblast differentiation. Indeed, p53−/− mice exhibited increased bone mass and bone formation, and p53−/− osteoblasts showed accelerated differentiation and up-regulated expression of osteogenic transcription factor Osterix.11 Moreover, the inhibition of p53 function mediated by oncogenic factor Mdm2 is a prerequisite for Runx2 activation and proper skeletal formation.12 In recent years, the Osterix-Cre transgenic mouse model, which targets osteoblast precursor cells during development, has been used to generate novel mouse models for osteosarcoma. When both p53 and pRb were deleted using the Osx-Cre mice, osteosarcoma occurred with almost 100% penetrance.13,14 Further analysis revealed that osteosarcoma development is likely to be initiated by the loss of p53 and potentiated by the loss of pRb.13,14
Cancer Stem Cell Hypothesis Author Manuscript
Heterogeneity is a defining feature of solid tumors. Several factors contribute to this heterogeneity, including genetic mutations, epigenetic changes, interactions with the microenvironment, cellular morphology, and proliferation and differentiation capacity.15,16 The cancer stem cell (CSC) hypothesis proposes that a heterogeneous tumor contains a hierarchal organization of cells in which only a small subset called CSCs are responsible for sustaining tumor growth.17 The first evidence of CSCs came from studies in acute myeloid leukemia (AML). In 1994, Lapidot et al. demonstrated that only a small percentage of AML cells were capable of generating leukemia when transplanted into immunodeficient mice.18 Since then, CSCs or CSC-like cells have been identified in several types of cancers, including breast19, brain20, colon21, prostate22, and bone23.
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CSCs are generally defined as tumor cells with stem-like characteristics that are responsible for sustaining tumor growth. Similar to normal stem cells, CSCs have self-renewal potential and differentiation capacity.17,24 The CSC division process is also similar to that of normal stem cells. CSCs can divide through either symmetric or asymmetric division, although primarily asymmetric division. During asymmetric division, CSCs divide to produce two daughter cells: an identical CSC with self-renewal capacity and a differentiated progenitor cell responsible for the majority of cell division (Figure 2).25 The exact origin of CSCs is uncertain. CSCs may arise from either normal stem cells or more differentiated cells that have acquired stem cell-like properties during malignant transformation.26
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In addition, evidence suggests that CSCs are resistant to cancer therapies such as chemotherapy and radiation. CSCs have several properties that make them resistant to traditional cancer therapies, including enhanced DNA repair ability, quiescence, increased expression of drug efflux transporters, and increased resistance to apoptosis.25 Traditional cancer therapies, therefore, may reduce the majority of the primary tumor but actually miss the small subset of CSCs. It is possible that CSCs are drug-resistant and consequently responsible for tumor recurrence and metastasis formation. Thus, the development of CSCtargeted therapies is important to prevent primary tumor recurrence and metastasis.
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Cancer Stem Cells in Osteosarcoma
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Recent studies have identified the existence of CSC-like cells in osteosarcoma. Gibbs et al. were the first to isolate a CSC subpopulation from nine biopsies of untreated osteosarcoma and the MG-63 osteosarcoma cell line using a sphere formation assay.23 The cells grew in spherical colonies, also called sarcospheres, when cultured with epidermal growth factor and basic fibroblast growth factor under serum starvation and anchorage-independent conditions. The sarcosphere-derived cells had increased expression of the stem cell markers Oct4 and Nanog compared with adherent cells. These sarcospheres were also shown to have the ability to self-renew through the formation of secondary spheres. Several other groups have since confirmed the capability of isolating CSCs expressing Oct4 and Nanog from osteosarcoma using sphere culture.27,28 In addition to Oct4 and Nanog, the stem cell transcription factor Sox2 has also been shown to be required for osteosarcoma selfrenewal.29 Using the MG-63 osteosarcoma cell line model, it has been shown that MG-63 spheres also have a higher resistance to chemotherapy drugs and a greater expression of DNA mismatch repair enzymes than adherent MG-63 cells.27 To date, sphere culture is the most widely used method for isolating osteosarcoma CSC-like cells. Sphere formation, however, can also occur within non-stem cell populations.30 Therefore, a more detailed analysis is needed to better characterize and identify osteosarcoma CSCs.
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CSC-like cells from osteosarcoma have also been isolated by fluorescent and magnetic cell sorting for CD133+ (prominin), CD117+ (c-kit), and Stro-1+ 2. Tirino et al. identified a small population of CD133+ cells in several human osteosarcoma cell lines with stem-like characteristics including a high proliferation rate and the ability to form spheres in culture.31 CD117+ Stro-1+ cells have been shown to have increased tumorigenicity when injected subcutaneously into mice, increased metastasis after orthotopic injections, and drug-resistant properties.32 Recently, Ying et al. used an inverse-lineage tracking strategy coupled with serial human-to-mouse xenotransplantation to identify osteosarcoma cells with CSC-like properties. They found that CD49f− CD133+ cells had CSC-like properties including selfrenewal and strong tumorigenicity that correlated with diminished osteogenic fate, while CD49f+ had limited tumorigenicity and more differentiated osteogenic features.33 High aldehyde dehydrogenase (ALDH) activity has also been used to identify CSC-like cells in osteosarcoma.2
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In addition to osteosarcoma, CSC-like cells have also been characterized in other sarcomas, including Ewing sarcoma and rhabdomyosarcoma, using sphere formation and marker analysis of CD133 and ALDH.26 Ewing sarcoma is the second most common malignant bone tumor and, similar to osteosarcoma, is associated with poor prognosis and low survival rates. Suva et al. isolated a subpopulation of CD133+ tumor cells from primary Ewing sarcoma tumors.34 They showed that these cells were able to initiate and sustain tumor growth through serial transplantation in immunodeficient mice. The CD133+ cells also expressed higher levels of stem cell markers Oct4 and Nanog than the CD133− cells. One major challenge in the osteosarcoma CSC field is to identify reliable and consensus CSC markers.2,26,35 One reason for the variability among different studies is that the markers expressed by CSC may differ among patients.36 Thus, putative osteosarcoma CSC
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markers need to be tested in a significant number of patients to determine how to assign those markers based on the type, subtype, or stage of cancer, as well as how to distinguish tumorigenic cells from nontumorigenic cells in different tumors. It will also be important to perform lineage tracing studies in order to determine the origin and characteristics of the CSCs that contribute to tumor growth and disease progression.
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Yang et al. recently reported what may be the first CSCs discovered in bone in a mouse model.37 Metachondromatosis is a benign cartilage-tumor syndrome caused by uncontrolled chondrocyte proliferation in humans, resulting from loss-of-function mutations in PTPN11. The authors deleted Ptpn11 in mice and, using lineage tracing studies, identified a population of progenitor cells in the perichondrium tissue layer that surrounds cartilage in long bones.37 These cells, similar to mesenchymal stem cells, can differentiate into several cell types including osteoblasts, chondrocytes, and adipocytes, in vitro. They have shown that Ptpn11 acts as a tumor suppressor in these CSC-like cells; therefore, the deletion of Ptpn11 initiates the formation of cartilage tumors.
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The transcriptional cofactor JAB1 is highly expressed in many different types of cancers, and has emerged as a novel player in tumorigenesis through its effects on cell proliferation, differentiation, survival, and cell cycle progression.38 Our laboratory is studying the potential role of JAB1 in the pathogenesis of osteosarcoma. We have confirmed that JAB1 is highly expressed in biopsy samples of osteosarcoma patients. In addition, JAB1 knockdown in the metastatic 143B osteosarcoma cell-line exhibits significantly reduced cell proliferation, colony formation, and motility, suggesting that down-regulating JAB1 expression in osteosarcoma reduces tumorigenesis. The underlying mechanisms of JAB1 involvement in tumor development, however, remain poorly understood. We have previously shown that Jab1 plays critical roles in the successive steps of skeletogenesis in vivo.39 Further elucidation of the crucial roles of Jab1 in osteoprogenitor cells during skeletal development may provide insights into the effect of JAB1 on key CSC properties, such as stem cell maintenance and renewal, and may eventually lead to the development of JAB1-targeted cancer therapies.
Conclusion
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The poor prognosis of osteosarcoma patients is related to drug resistance, and osteosarcoma cancer stem cells are proposed to be at least partly responsible for this drug resistance. The development of CSC-targeted therapy, therefore, can potentially prevent tumor recurrence and increase survival rates of osteosarcoma patients. However, CSC research is still in its infancy, especially for osteosarcoma and other bone cancers, and many challenges still remain. Future research is needed to determine an optimal method for osteosarcoma stem cell identification and isolation, and to further understand the mechanisms of CSC selfrenewal, metastasis, and drug resistance.
Acknowledgements We thank Valerie Schmedlen for editorial assistance.
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Figure 1.
Disruption in osteoblast differentiation may lead to the development of osteosarcoma. (A) Progression of normal osteogenic differentiation. Mesenchymal stem cells (MSCs) are pluripotent cells that can differentiate into many cell types. During a tightly regulated process controlling the balance between cell proliferation and cell differentiation, MSCs can eventually differentiate into mature osteoblasts. (B) When osteogenic differentiation is disrupted, osteosarcoma precursors may form, which can lead to osteosarcoma. This disruption may be caused by genetic changes, such as, the inactivation of tumor suppressors p53 and Rb, and/or epigenetic changes. These changes can lead to increased proliferation and decreased differentiation of cancer cells. (adapted from [10])
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Figure 2.
Cancer stem cell (CSC) hypothesis model. CSCs divide into two cells: 1) an identical CSC which self-renews and maintains a pool of CSCs (white balls), and 2) a differentiated progenitor cell that continues to proliferate generating heterogeneous cancer cells comprising the bulk of the tumor (grey balls). (adapted from [40])
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