Review Article Overcoming Therapeutic Resistance of Bone ... · Overview of the Molecular Mechanisms and Therapeutic Targets for Bone Sarcoma Stem Cells TomohiroFujiwara 1,2 andToshifumiOzaki

Review ArticleOvercoming Therapeutic Resistance of Bone SarcomasOverview of the Molecular Mechanisms and TherapeuticTargets for Bone Sarcoma Stem Cells

Tomohiro Fujiwara12 and Toshifumi Ozaki1

1Department of Orthopedic Surgery Okayama University Graduate School of Medicine Dentistry and Pharmaceutical SciencesOkayama 7008558 Japan2Department of Intelligent Orthopaedic System Okayama University Graduate School ofMedicine Dentistry and Pharmaceutical Sciences Okayama 7008558 Japan

Correspondence should be addressed to Tomohiro Fujiwara tomomedvngmailcom

Received 3 July 2016 Revised 31 October 2016 Accepted 16 November 2016

Academic Editor James Adjaye

Copyright copy 2016 T Fujiwara and T Ozaki This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Bone sarcomas are heterogeneousmalignant tumors that exhibit clinical histological andmolecular heterogeneity Recent progressin their multimodal treatment has gradually improved patient prognosis however drug resistance and distant metastasis remainunresolved clinical problems Recent investigations have suggested the existence of cancer stem-like cells (CSCs) in bone sarcomaswhich represent a subpopulation of tumor cells with high tumor-forming ability The hallmarks of CSCs include tumor- andmetastasis-forming potential and drug resistance which are responsible for poor prognoses of bone sarcoma patients Thereforeelucidation of the molecular mechanisms of CSCs and identification of therapeutic targets could contribute to novel treatmentstrategies for bone sarcomas and improve patient prognosis This paper provides an overview of the accumulating knowledge onbone sarcoma stem cells and preclinical analyses to overcome their lethal phenotypes in addition to a discussion of their potentialfor novel therapeutics for bone sarcomas

1 Introduction

Bone sarcomas are a heterogeneous group of malignant bonetumors characterized by various degrees of mesenchymaldifferentiation Since their origin has not been identifiedbone sarcoma classification is based on morphological find-ings such as cell type architecture and matrix productionThe World Health Organization (WHO) system is generallyaccepted as the basis for bone sarcoma classification [1] Bonesarcomas constitute 02 of all malignancies in adults andapproximately 5 of childhood malignancies as determinedby the Surveillance Epidemiology and End Results (SEER)study Cancer registry data with histological stratificationindicate that osteosarcoma is the most common primarybone sarcoma constituting approximately 35 followed bychondrosarcoma with 25 and Ewing sarcoma with 16 [2]

Osteosarcoma is the most common primary malig-nant tumor of bone with a peak incidence in adolescentsand young adults With combined treatment (neoadjuvantchemotherapy surgery and adjuvant chemotherapy) the 5-year survival rate for patients with no metastatic disease atdiagnosis is 60ndash80 [3ndash5] However for poor responders tochemotherapy and patients withmetastatic disease outcomesare far worse at lt50 and lt30 survival respectively [3 6]The survival rate has hardly improved for 20 years despitemultiple clinical trials Likewise the current chemotherapyprotocols used to treat Ewing sarcoma the secondmost com-mon sarcoma of bone in children and young adults includevarious combinations of the following six drugs doxorubicincyclophosphamide vincristine actinomycin-D ifosfamideand etoposide Biologically Ewing sarcoma is characterizedby recurrent balanced translocations involving the EWSR1

Hindawi Publishing CorporationStem Cells InternationalVolume 2016 Article ID 2603092 13 pageshttpdxdoiorg10115520162603092

2 Stem Cells International

gene and amember of the ETS family of transcription factorsmost commonly FLI-1 [7] Although multidisciplinary careincorporating advances in diagnosis surgery chemotherapyand radiation has substantially improved the survival rateof patients with localized Ewing sarcoma to nearly 70 [8]survival in a metastatic or recurrent disease setting remainsextremely low at lt20 Chondrosarcoma a malignantgroup of cartilaginous matrix-producing neoplasms typicallyoccurring in the fifth to seventh decades of life is generallyresistant to chemotherapy and radiotherapy while Ewing sar-coma is relatively sensitive [1] Its prognosis depends largelyon the histological grade and treatment is mostly limited tosurgical resection [9]

The clinical outcomes of these bone sarcomas haveplateaued for the last 10 years Considering the characteristicsand heterogeneity of bone sarcomas it is possible that asubset of tumor cellsmight resist various stresses and producerecurrence ormetastasis which corresponds to the hallmarksof cancer stem-like cells (CSCs) Indeed there are no fewerbone sarcoma cases involving metastases long after initialtreatments [10] Although targeted therapy for bone sarcomastem cells has not been available several preclinical trials havebeen reported which might improve patient prognosis Thispaper provides an overview of the accumulating knowledgeon bone sarcoma stem cells and preclinical analyses toovercome their lethal phenotypes

2 Cancer Stem Cell Hypothesis inBone Sarcomas

The cancer stem cell hypothesis is based on the observationthat not all cells in tumors are equal [11] It proposes thatthere is a small subpopulation of cells within a heterogeneoustumor that are responsible for forming the bulk of the tumor[12 13] These cells are similar to normal stem cells and mayarise from the transformation of stem cells or the dedifferen-tiation of nonstem cells [14] The common consensus is thatthey are able to self-renew and differentiate into all of thecells within a tumor [12] The first evidence of the existenceof CSCs was reported in hematological malignancies [11]with these cells being characterized as the CD34+CD38minusfraction [15] CSCs have now been isolated from varioushuman solid tumors including bone sarcomas [13] The firstdemonstration of the existence of bone sarcoma stem cellswas achieved by Gibbs et al in 2005 who showed thatosteosarcoma and chondrosarcoma cells include a subpopu-lation of cells that are capable of growing in spheres and havethe properties of self-renewability and multipotency [16]Thereafter several CSC markers that are common to othermalignant diseases as well as unique to bone sarcomas havebeen identified (Figure 1) Recent investigation has focusedon themolecularmechanisms underlying bone sarcoma stemcells and therapeutic testing using preclinical models

3 Characterization of Bone SarcomaStem Cells

Since the first identification of osteosarcoma stem cells byGibbs et al in 2015 various reports on osteosarcoma and

Ewing sarcoma stem cell have been reported while beinglimited for chondrosarcoma Most of these reports havedocumented the conventional CSC markers such as CD133ALDH and side population Notably the mesenchymalstem cell markers such as CD117Stro-1 and CD273 havebeen included which are the unique characteristic of bonesarcoma stem cells (Figure 1 Table 1) However there is noclear consensus on the definite marker that characterizesbone sarcoma stem cells and discussions of several markerssuch as side population have caused controversies in theliterature

31 Sarcosphere (Osteosarcoma Ewing Sarcoma and Chon-drosarcoma) Gibbs et al first reported that several osteosar-coma cell lines established from biopsy specimens andMG63OS cell line formed spheres at a frequency of 01ndash1 in ananchorage-independent environment which could form sec-ondary spheres after the dissociation of single cells [16]Wanget al also identified sphere formation from OS99-1 MG63HuO9 and SaOS2 [17] and Wilson et al found similaritiesbetween human and canine osteosarcoma cell lines in termsof forming spheres [18] Fujii et al further identified thatMG63 spheres showed resistance to doxorubicin and cisplatinand increased expression of the DNA repair enzyme genesMLH1 andMSH2 indicating that a DNA repair inhibitor hadthe potential to enhance the efficacy of chemotherapy [19]

Human Ewing sarcoma cell line also formed spheres at alow frequency rate which expressed higher levels of Oct34Nanog STAT3 Sox2 Sox10 and EWS-FLI1 and showedhigher drug resistance similarly to OS cells [19] Similarlychondrosarcoma cells that originated from patient biopsiesformed spherical colonies which displayed Stro-1 CD44 andCD105 the mesenchymal stem cell markers [16] HoweverLeuchte et al demonstrated that sphere formation is nota reliable method to enrich CSCs of Ewing sarcoma sincethese spheres did not continuously self-renew by secondarysphere formation and sphere culture did not enhance thetumorigenicity in vivo [20] Therefore the sphere modelmight be inadequate to reflect CSC properties but at leastuseful for preclinical in vitro testing of novel therapies

32 CD133 (Osteosarcoma Ewing Sarcoma and Chondrosar-coma) CD133 first recognized as a novel antigen on CD34+progenitor hematopoietic stem cells is a glycoprotein with afive-transmembrane-domain protein encoded by the PROM1gene [21ndash23] Tirino et al were the first to demonstrate thatCD133+ osteosarcoma cells possessed CSC phenotypes Theyshowed that SaOS2 MG-63 and U2OS cell lines containeda small fraction of CD133+ cells ranging from 3 to 5which showed the following phenotypes high proliferationrate cell cycle detection in G

2M phase positivity for Ki-

67 formation of spheres and inclusion of a small subset ofSP cells (097) [24] They further identified that all humanosteosarcoma and chondrosarcoma samples contained asmall population ofCD133+ cells [25]Moreover CD133+ cellsof two stabilized cell lines from clinical samples (50ndash78)also showed self-renewability sphere formation adipogenicand osteogenic differentiation high expression of stemnessgenes and tumorigenicity [25]

Stem Cells International 3

Enoxacinb

MST312a BRM270a

HIF2PUTa

TSSC3a Bufalina

Salinomycina

TRBP2b

Synthetic miR-143 145ab

miR-1247a

LNAa

miR-133aa

EWS-FLI-1

BMP2a

Wnta

Notcha

CD57bCD271a

Sox2aOct34abc

CD47a

ALDHab

CD248a

Stro-1aCD117c-kita

CD133abcd

Telomerasea MC1742a

YK-4-209b

NF-120581Ba

TGF-120573a

TNF-120572a

(a) Osteosarcoma (c) Chondrosarcoma (b) Ewing sarcoma

Figure 1 Overview of bone sarcoma stem cells Although there is no consensus on the definitive marker a wide range of CSCmarkers (black)and the molecular mechanisms underlying CSC phenotypes (blue) have been documented for each sarcoma Several anti-CSC compounds(red) have been preclinically tried to inhibit CSC phenotypes a osteosarcoma b Ewing sarcoma c chondrosarcoma

Several molecules have been used in combination withCD133 expression as CSC markers For example Veselska etal identified the cells expressing nestin in 18 osteosarcomaprimary samples Among 4 of the 18 stabilized cell lines 3contained nestin+CD133+ cells at a frequency of 11ndash100[26] In addition He et al added CD44 to their analysis andfound that the CD133+CD44+ fraction was more aggressiveregarding sphere formationmigration and invasiveness thanits counterparts and it showed the strongest potential fortumorigenicity and lung metastasis [27] On the other handYing et al identified that CD49fminus fraction showed asymmet-ric division and contained a CD133+ fraction that increasedin culture over time Subsequently CD49fminusCD133+ fractionwas revealed to show strong tumorigenicity correlated withan impaired osteogenic fate [28]

CD133+ fraction was also identified in Ewing sarcomacells Suva et al isolated CD133+ cells from three surgically

resected tumors and revealed 4ndash8 of bulk cell popula-tions These CD133+ cells displayed higher tumorigenicityspherogenic potential multipotency and high expressionlevels of Oct-4 and Nanog [29] On the other hand Jiang etal reported that CD133+ cells were identified in only 4 of 48primary tumor specimens [30] Among these four patientswith CD133+ two were drug-resistant but the others wereevent-free survivors Moreover no differences were observedin the drug resistance or tumorigenicity between CD133+and CD133minus cells except for the STA-ET-82 cell line [30]Therefore their study suggests that CD133 is significant as aCSC marker only in some cases of Ewing sarcoma

33 Side Population Cells (Osteosarcoma Ewing Sarcoma andChondrosarcoma) A subpopulation that effluxes the DNA-binding dye Hoechst 33342 out of the cell membrane throughan ATP-binding cassette (ABC) transporter was recognized


Table 1 Markers of bone sarcoma stem cells

Subtype Marker Function clinical relevance Reference

Osteosarcoma

Sarcosphere Drug resistance overexpressing Oct34Nanog and Stat3 [16ndash19]

CD133Sphere formation multipotency

tumorigenicity self-renewal inclusion of SPcells overexpressing Oct34 and Nanog

[24ndash26]

CD49fminus Sphere formation migration invasiontumorigenicity lung metastasis [28]

CD44 Sphere formation migration invasiontumorigenicity lung metastasis [27]

CD117Stro-1Drug resistance invasion metastasis

tumorigenicity self-renewal overexpressingABCG2 and CXCR4

[25 41]

SP Drug resistance self-renewal tumorigenicity [34 35]CD248 Tumorigenicity invasion [40]

ALDH Proliferation tumorigenicity overexpressingOct34 Nanog Sox2 and Stat3 [47 48]

CBX3 and ABCA5 Highly expressed in spheres [49]

CD47 Invasion blockage of macrophagephagocytosis prognostic value [52]

CD271Self-renewal differentiation drug resistancetumorigenicity overexpressing Oct34 Nanog

Stat3 Bcl-2 and ABCG2[54]

Oct34 Tumorigenicity self-renewal [57 58]

Sox2 Soft agar growth migration invasiontumorigenicity reduced Wnt signaling [59]

Ewing sarcoma

SarcosphereDrug resistance overexpressing Oc34 Nanog

Stat3 Sox2 Sox 10 and EWS-FLI1 fail toself-renew and enhance tumorigenicity

[19 20]


tumorigenicity overexpressing Oct34 andNanog no difference in drug resistance and

tumorigenicity

[29 30]

CD57 Migration invasion multipotencytumorigenicity no correlation with CD133 [55]

SP Drug resistance clonogenicity invasionasymmetric division [33 36 37]

ALDH Clonogenicity sphere formationtumorigenicity drug resistance [46]

ChondrosarcomaSarcosphere Multipotency overexpressing Oct34 Nanog

and Stat3 expressing Stro-1 CD44 and CD105 [16]

CD133 Tumorigenicity [25]SP NA [34]

SP side population ALDH aldehyde dehydrogenase NA not available

as a stem cell population in analyses of hematopoieticstem cells [31 32] This cell population expressing the ABCtransporter is defined as side population (SP) cells whichare distinguished from cells of the other population calledmain population (MP) cells [33] Wu et al reported thatSP cells were identified in five surgically excised osteosar-coma samples and two chondrosarcoma samples and showedhigher tumorigenicity than MP cells [34] SP cells werenegative for two cases of grade 1 chondrosarcoma SP cellswere also identified at a frequency of 12 from Ewing cell

line SK-ES-1 cells SP cells regenerated both SP and non-SPcells and showed higher clonogenicity drug resistance andinvasiveness than non-SP cells [35]

However SP cells were detected in only one osteosarcomaand Ewing sarcoma cell line respectively and tumorigenicityof these cells was controversial [33 36] Therefore furtheranalyses including serial transplantation experiments areneeded to determine the significance of SP cells as a bone sar-coma stem cell fraction [37] Moreover tumor cells resistantto Hoechst 33342 dye do not necessarily show tumorigenicity


and metastatic ability [38] and cytometry gating used toisolate SP cells lacks the consistency of gating strategiesused in marker staining [39] indicating the controversyof SP cells as a CSC fraction Sun et al confirmed thatprimary osteosarcoma specimens contained approximately39 fraction of SP cells and identified the high expressionof CD248 (endosialin) and other stem cell markers suchas Oct34 Nanog nestin and CD133 [40] SPCD248highcells showed strong tumorigenicity and invasiveness [40]indicating that CD248 is a potential therapeutic target

34 CD117 and Stro-1 (Osteosarcoma) CD117 and Stro-1 areexpressed in mesenchymal stem cells [41] CD117+Stro-1+cells of osteosarcoma showed CSC phenotypes with highinvasiveness and drug resistance Adhikari et al foundthat the CD117+Stro-1+ fraction of murine cell lines haddifferentiation ability highly expressed CXCR4 and ABCG2and showed higher drug resistance tumorigenicity andmetastatic ability than CD117minusStro-1minus cells [41] These prop-erties were validated in human osteosarcoma cell linessuggesting that CD117+Stro-1+ cells possess CSC propertiesHowever the reliability of CD117+Stro-1+ in clinical samplesremains controversial Tirino et al reported that all clinicalsamples in their analysis were negative for CD117 indicatingthat further investigation and discussion are necessary toconfirm its potential as a CSC marker [25]

35 ALDH (Osteosarcoma and Ewing Sarcoma) ALDH is adetoxifying enzyme responsible for the oxidation of intracel-lular aldehydes [42] which has been reported to play a rolein the early differentiation of stem cells in the oxidation ofretinol to retinoic acid [43] High ALDH activity has beenobserved in murine and human hematopoietic and neuralstem and progenitor cells [44 45] and the evaluation ofALDH activity has been a useful approach in the isolation ofCSCs in several tumors [45 46] Wang et al reported thathighly aggressive OS99-1 contained cells with high ALDHactivity (ALDHbr cells) at a rate of 451 while the corre-sponding rates were 18 in HuO9 16 in SaOS2 and 06in MG63 [47] ALDHbr cells from a xenograft showed higherproliferation rate clone formation expression of Oct34ANanog and Sox2 and tumorigenicity than ALDHlo cellsHonoki et al reported the similar results in spheres whichdisplayed stronger drug resistance than monolayer adherentcells [48] Awad et al demonstrated that Ewing sarcomaALDHhigh cells were enriched for clonogenicity sphere for-mation the expression of Oct34 Bmi-1 and Nanog drugresistance and tumorigenicity [46] Notably ALDHhigh cellswere resistant to doxorubicin but sensitive to YK-4-279 asmall-molecule inhibitor of EWS-FLI1 [46] YK-4-279 blocksoncogenic activity of EWS-FLI1 by blocking its interactionwith RNA helicase A (RHA) and induces apoptosis in Ewingsarcoma cells both in vitro and in vivo However ALDHhigh

activity was not investigated in human specimens thereforefuture studies will be needed to evaluate the significance ofALDHhigh cells and their clinicopathological correlations inEwing sarcoma

36 CBX3 andABCA5 (Osteosarcoma) Saini et al confirmedthat spheres from clinical specimens showed self-renewalclonogenic and tumorigenic potential and identified thatthey had lower expression levels of CD326 CD24 CD44and higher ABCG2 [49] In addition proteomic and tran-scriptome analyses revealed high expression of chromoboxprotein homolog 3 (CBX3) and ABCA5 respectively whichwas significantly higher in osteosarcoma than in primaryosteoblast

37 CD47 (Osteosarcoma) CD47 is a transmembrane proteinthat acts as a self-signal on normal cells by inhibitingmacrophage phagocytosis [50] and high expression of CD47is a poor prognostic factor [51] Xu et al identified that CD47protein was highly expressed in tumor tissue compared withthat in normal controls and that the majority of CD44+ cellsexpressed CD47 (80ndash99) [52] Interestingly CD47 block-age increased the macrophage phagocytosis of tumor cellsindicating that this could be an effective immunotherapeuticapproach

38 CD271 (Osteosarcoma) CD271 is one of the cell surfacemarkers of bonemarrowMSCs andwas identified as amarkerof melanoma CSCs [53] Tian et al found that CD271 wasexpressed in tumor tissues (0ndash29) and in a small popu-lation in osteosarcoma cell lines (54ndash97) CD271+ cellsdisplayed the properties of self-renewal differentiation drugresistance and tumorigenicity and showed higher expressionofOct34Nanog Stat3 Bcl-2 andABCG2 thanCD271minus cells[54]

39 CD57 (Ewing Sarcoma) Wahl et al investigated whetherCD57 (HNK-1) a surface marker for migrating and prolif-erating neural crest cells could be a CSC marker in Ewingsarcoma [55]TheCD57 expression level positively correlatedwith sphere formation CD57high cells were adhesive invasiveand tumorigenic compared with CD57low cells However theaggressiveness of CD57high cells could not be solely attributedto the coexpression of CD133 since CD57high VH-64 cells didnot express CD133 while CD57high WE-68 cells were alsopositive for CD133 [55]

4 Signaling Pathways Activated in BoneSarcoma Stem Cells

Since most of the CSC markers summarized in the previoussection are expressed on the normal cells and tissues acomprehensive understanding of the tumor-specific molec-ular pathways underlying CSCs is needed for therapeuticapplication Analyses of the signaling pathways of CSCshave been performed with the isolation technique based onthe CSC markers in Table 1 Diverse molecular pathwayssuch as self-renewal and epigenetic alterations have beendocumented which are further investigated as therapeuticexperiments (Figure 1 Table 2)

41 Self-Renewal Marker Genes (Oct34 Nanog and Sox2)The transcription factors Oct34 Sox2 and their target


Table 2 Regulators of bone sarcoma stem cells

Subtype Category Regulators Target CSC Function clinical relevance Reference

Osteosarcoma Signaling pathways

MAPK 3AB-OS Self-renewal tumorigenicity [60 61]

Wnt120573-catenin Sarcosphere SP Tumorigenicity drug resistanceinvasion [64 65]

Notch ALDH Oxidative stress migrationinvasion VEGF expression [69]

TGF-120573 Sarcosphere Dedifferentiation clonogenicity [70]TNF-120572 AX Tumorigenesis differentiation [71]

BMP2 ALDH Tumorigenesis stem-cell markerdifferentiation marker [72]

Osteosarcoma Epigenetic regulators

miR-29b 3AB-OS Sphere clonogenicity drugresistance [76]

miR-133a CD133 Invasion metastasis prognosis [77]

miR-143 CD133ALDH Drug resistance autophagyprognosis [78]

miR-1247 CD117Stro-1 Sphere proliferation [79]

HIF2PUT CD133 Sphere proliferation migrationalteration of HIF2-120572mRNA [82]

Telomerase Sarcosphere Sphere invasion drug resistancetumorigenicity [85]

TSSC3 Sarcosphere Sphere clonogenicity apoptosisregulate Oct34 Nanog and Sox2 [86]

MLH1 MSH2 Sarcosphere Drug resistance [19]

Ewing sarcoma Epigenetic regulatorsTARBP2 CD133 Tumorigenicity CD133+ frequency [81]miR-145 CD133 Tumorigenicity [80]

miR-143 -145 CD133 Clonogenicity tumorigenicity [81]

Nanog are known as key regulators of pluripotency [56]Aberrant expression of Oct34 and Nanog has also beensuggested to fulfill an oncogenic role in tumorigenesis andthe development of CSCs [57 58] Levings et al found thatosteosarcoma cells derived from biopsy samples containeda small population of self-renewing spherical clones thatshowed significant increases of Oct-4 and Nanog expressionTumorigenic OS521 cells were engineered to activate anOct-4 promoterGFP reporter and the GFP+ cells were atleast 100-fold more tumorigenic and metastatic capable offorming tumors at less than 300 cells whereas only 1 of 8mice developed a tumor at 3000 cells in the GFP-depletedgroup [57 58] OS521Oct4-pGFP+ cells were capable of self-renewal in several passages forming heterogeneous tumorsfor Oct-4GFP expression Basu-Roy et al found that Sox2mRNA and protein were highly expressed in human andmurine osteosarcoma cell lines as well as osteosarcoma tissuesamples at variably high levels [59] Sox2 depletion by shRNAdecreased colony formation in soft agar migration invasionand tumorigenicity High Sox2 expression was accompaniedby reduced Wnt signaling while the activation of Wntsignaling resulted in low Sox2 expression [59] suggesting thatthe activation of Wnt signaling antagonizes the effect of Sox2in maintaining osteosarcoma cells

42 MAPK Wnt120573-Catenin and Notch Pathway Themitogen-activated protein kinase (MAPK) pathway

is frequently activated in human cancers includingosteosarcoma [60] Gemei et al performed proteomic anal-ysis using stem-like 3AB-OS cells and determined that theERKMAPK pathway is associated with tumorigenicity andself-renewability [61]

TheWnt signal transduction pathway coordinatesmyriadactivities from development and differentiation to prolifer-ation and tumorigenesis [62] Aberrant Wnt signaling hasbeen reported in various tumors and shown to be associ-ated with CSC activity [63] Martins-Neves et al reportedthat tumorigenic osteosarcoma spheres overexpressed SOX2and KLF4 and showed specific activation of Wnt120573-cateninsignaling [64] In addition Yi et al confirmed that SP cellswere contained in osteosarcoma samples and 120573-catenin andcyclin D1 were highly upregulated in them indicating thatthis pathway is a potential target of novel anticancer drugsfor osteosarcoma stem cells [65]

Notch signaling plays a key role in the normal develop-ment of many tissues and cell types through diverse effectson cell fate stem cell renewal differentiation survival andproliferation [66] This signaling pathway functions as anoncogene or a tumor suppressor depending on the cellularcontext [67] and is associated with osteosarcoma and Ewingsarcoma [68] Mu et al reported that ALDH activity in highlymetastatic K7M2 cells is reduced by Notch inhibition and isalso associated with increased resistance to oxidative stress


migration invasion and VEGF expression suggesting thatALDH activity may be regulated by Notch signaling [69]

43 TGF-120573 TNF-120572 and BMP-2 Transforming growthfactor-beta (TGF-120573) is a pleiotropic cytokine that helpsto maintain homeostasis limits the growth of epithelialendothelial neuronal and hematopoietic cell lineages andacts as a mediator on tumors to promote further tumorexpansion Zhang et al reported that TGF-1205731 signalingand a hypoxic environment induced CSC phenotypes ina non-CSC population and that the blockage of TGF-1205731signaling inhibited the dedifferentiation and clonogenicity ofosteosarcoma cells and reduced CSC self-renewal capacitysuggesting that CSCs may be yielded from differentiated cells[70]

Tumor necrosis factor-120572 (TNF120572) is an inflammatorycytokine produced by macrophagesmonocytes during acuteinflammation and is responsible for a range of signalingevents leading to necrosis or apoptosis In addition Moriet al reported that TNF120572 is required for the tumorigenesisof osteosarcoma Lethal tumorigenesis of AX osteosarcomacells was completely abrogated in TNF120572-deficient mice andIL-1aIL-1b doubly deficient mice which occurred throughERK activation [71]

Bone morphogenetic proteins (BMPs) are members ofthe TGF-120573 superfamily which play pivotal roles in not onlybone and cartilage formation but also cell proliferationapoptosis differentiation and tumorigenesis Wang et alevaluated the effect of BMP2 on ALDHbr OS cells anddetermined that it suppresses tumor growth by reducing theexpression of Oct34 Nanog and Sox-2 genes and inducingthe differentiation markers Runx-2 and collagen type I [72]

44 Epigenetic Regulators in CSCs

441MicroRNA (miR-29b 133a 143 145 and 1247) miRNAsare small regulatory RNA molecules that modulate theexpression of their target genes and play important rolesin a variety of physiological processes such as develop-ment differentiation cell proliferation apoptosis and stressresponses [73] In recent years many miRNAs have beeninvestigated in various malignant diseases [74] Deregulationof the expression of miRNAs has been shown to contribute tocancer development through various mechanisms includingdeletions amplifications ormutations involvingmiRNA lociepigenetic silencing and the dysregulation of transcriptionfactors that target specific miRNAs [75] Recently miRNAshave been focused on as a novel approach for regulating thephenotypes of CSCs of bone sarcomas Di Fiore et al iden-tified that miR-29b-1 suppressed the stemness properties of3AB-OS CSCs including spherecolony formation and drugresistance but did not their invasive and migratory abilities[76] Our microarray analysis revealed upregulated miR-133aexpression levels in the CD133high fraction High expressionlevels of miR-133a and low expression levels of its target geneswere significantly correlated with clinical prognosis and thesilencing of miR-133a contributed to cell invasion and lungmetastasis [77] Zhou et al found that tumor-suppressive

miR-143 was downregulated in CD133+ALDH+ cells andassociated with drug resistance and autophagy From amicroarray analysis with CD117+Stro-1+ and CD117minusStro-1minuscells [78] Zhao et al found significant downregulation ofmiR-1247 which targets MAP3K9 promoting OS prolifera-tion and sphere formation [79]

Riggi et al identified Ewing sarcoma stem cells usinghuman pediatric MSCs (hpMSCs) They demonstrated thathpMSCs expressing EWS-FLI-1 (hpMSCEWS-FLI-1) generatea CD133+ subpopulation displaying CSC phenotypes [80]Interestingly EWS-FLI-1 induced the expression of Sox2Oct-4 and Nanog through miR-145 repression and theyfunction in a feedback loop with their common target geneSox2 which regulates the differentiation and tumorigenicityof Ewing sarcoma cells In addition De Vito et al addedthe molecular mechanisms underlying Ewing sarcoma stemcells They found that TARBP2 (TAR RNA-binding protein2) which forms part of the Dicer-1 complex was suppressedin CD133+ Ewing sarcoma cells as well as hpMSCEWS-FLI-1and enoxacin which augments TARBP2 function inhibitedtumor growth through the restoration of miRNAmaturation[81] Similarly the systemic injection of 30 120583g of syntheticTARBP2-dependentmiR-143 ormiR-145 showed a significantreduction of tumor volume suggesting that CSC phenotypesof Ewing sarcoma correlatewith the deregulation of TARBP2-dependent miRNA expression [81]

442 Long Noncoding RNA (HIF2PUT) Among the var-ious types of noncoding RNA miRNAs have been mostextensively studied and their role in carcinogenesis has beenestablished but several long noncoding RNAs (lncRNAs)with functional involvement in malignant diseases have alsobeen identified Wang et al investigated the function ofhypoxia-inducible factor-2120572 (HIF-2120572) promoter upstreamtranscript (HIF2PUT) in osteosarcoma stem cells The pro-portion of CD133+ cells decreased and inhibition of sphereformation proliferation and migration was decreased bythe overexpression of HIF2PUT while the knockdown ofHIF2PUT showed the opposite function via alteration ofHIF-2120572mRNA expression [82]

443 Telomerase Human telomerase is a reverse transcrip-tase composed of a catalytic component telomerase reversetranscriptase (TERT) and a telomerase RNA component(TERC) [83] In most normal human somatic cells telom-erase activity is undetectable while stemprogenitor cellsexpress telomerase and its activity is also detected in mostmalignant cancers [84] Yu et al identified high telomeraseactivity in sphere-derived osteosarcoma cells (TEL+) andTEL+ cells showed increased sphere and tumor-propagatingcapacity invasiveness metastatic activity and drug resis-tance Treatment with MST312 a telomerase inhibitor wasalso shown to target TEL+ cells and prevent the tumorigenic-ity of osteosarcoma cells [85]

444 TSSC3 Huang et al found that overexpression ofthe imprinted gene TSSC3 (tumor-suppressing STF cDNA3) an apoptosis-related gene was associated with growth


Table 3 Preclinical trials of novel agents targeting bone sarcoma stem cells

Subtype Agents Function Target CSC Mechanism Reference

Osteosarcoma

LY294002 PIK3 inhibitor SarcosphereIncrease the number of cells in G

0G1

phase and induction of apoptosis viaactivation of caspase-3 caspase-9 and

PARP

[87]

BRM270 NF-120581B inhibitor CD133Induce IL-6 mediated apoptosis inosteosarcoma CD133+ cells via

downregulation of chromatin SMC2[91]

MC1742 HDAC inhibitor SarcosphereInhibit sphere growth of osteosarcoma

and Ewing sarcoma by apoptosisinduction with increased acetyl-H3

and acetyl-tubulin levels

[95]

LNA-133a miR-133a inhibitor CD133Inhibit invasion and metastasis of

osteosarcoma CD133+ cells via severaltarget genes including ANXA2

[77]

Salinomycin Antibacterial agent Sarcosphere Impair Wnt120573-catenin signaling bydegradation of 120573-catenin [99]

Ap-SAL-NP Nanoparticle CD133Selectively kill CD133+ cells bysalinomycin-loaded PEGylated

polynanoparticles conjugated withCD133 aptamer

[89]

Bufalin Unknown SarcosphereInduce shrinkage of tumor spheres via

activation of caspase-3 anddownregulate stem cell markers

targeting miR-148

[100]

Ewing sarcoma

YK-4-209 EWS-FLI1 inhibitor ALDH Block RNA helicase A (RHA) bindingto EWS-FLI1 [46]

Enoxacin Antibacterial agent CD133Augment TARBP2 expression which isrepressed in CD133+ Ewing cells andreduce CD133+ subpopulation through

restoration of miRNA expression

[81]

miR-143145 Synthetic miRNA CD133 Repress the expression of target genesOct34 Sox2 as well as EWS-FLI1 [81]

inhibition and apoptotic induction in osteosarcoma theythen further analyzed the effect of this on osteosarcoma stemcells [86] The expression of TSSC3 was low in osteosarcomaspheres and the overexpression of TSSC3 downregulatedthe expression of the stem cell markers Oct34 Nanogand Sox2 decreased the clone formation rate and inducedapoptosis suggesting that TSSC3 plays a suppressive role inosteosarcoma spheres [86]

5 Therapeutic Targeting of Bone SarcomaStem Cells

Preclinical studies of the therapeutic application to targetbone sarcoma CSCs have now been performed on the basisof studies concerning the molecular biology underlyingCSCs Strategies include using the molecular target druginhibiting NF-120581B PI3K HDAC or a ldquonext generationrdquonucleic-acid therapeutics (Figure 1 Table 3) The infor-mation on these compounds is partly available from thehttpsClinicalTrialsgov website Although there are littlecompounds that have been approved for clinical use severalcandidates have already been tested in promising trials

51 Inhibitor of PI3K Signaling Pathway The phosphatidyli-nositol 3-kinase (PI3K) signaling pathway has inherentoncogenic potential and is involved in CSC biology inseveral malignancies including breast colon pancreas brainand bladder [87] Gong et al investigated the efficacy ofLY2940002 a PIK3 inhibitor that prevents the phosphory-lation of protein kinase B and revealed that this compoundincreased the number of cells in theG

0G1phase and induced

apoptosis via the activation of caspase-3 caspase-9 andPARPin osteosarcoma stem cells [88] Preclinical experimentswith in vivo models have not been completed although aphase I study of SF1126 a novel inhibitor of PI3 kinase andmTOR that includes an activemoiety consisting of LY294002for patients with relapsed or refractory neuroblastoma isreportedly ongoing [89]

52 Inhibitor of NF-120581B Signaling Pathway Nuclear factor 120581B(NF-120581B) comprises a family of transcription factors involvedin the regulation of various biological responses NF-120581B playsan important role in the regulation of immune responsesand inflammation but accumulated evidence has shown thatit also plays a major role in oncogenesis There is evidence


that NF-120581B-regulated gene products play a major role ininhibiting apoptosis in leukemic stem cells [90]Mongre et alinvestigated the efficacy of BRM270 a well-known traditionalChinese medicine against stem-like cancer-initiating cellsthis compound selectively inhibited NF-120581B transcriptionalactivity resulting in decreased expression of interleukin-6 which is a cytokine associated with metastasis BRM270induced IL-6-mediated apoptosis in CD133+ osteosarcomastem cells via the downregulation of chromatin SMC2 [91]However at present there is no information on its clinicaluse or trials in CSC therapy [92]

53 HDAC Inhibitor Epigenetics is defined as heritablechanges in gene expression that are not accompanied bychanges in the DNA sequence it includes DNAmethylationhistone modifications nucleosome positioning and non-coding RNAs Histone deacetylase (HDAC) inhibitors havebeen reported to suppress CSC phenotypes in solid malig-nancies [93 94] Di Pompo et al tested HDAC inhibitorson sarcospheres of osteosarcoma Ewing sarcoma and rhab-domyosarcoma and reported that MC1742 and MC2625inhibited sphere growth by inducing apoptosis with increasedacetyl-H3 and acetyl-tubulin levels [95] However in vivopreclinical sarcoma models have not been yet establishedAlthough there is no information on clinical trials for thesecompounds several epigenetic drugs including azacitidinedecitabine vorinostat and romidepsin have already receivedFDA approval and undergone clinical trials for osteosarcomaEwing sarcoma and other soft-tissue sarcomas [92]

54 MicroRNA Therapeutics miRNAs provide new thera-peutic targets for many diseases and recent progress in thedevelopment of effective strategies to adjust miRNA dysreg-ulation has indicated their potential for clinical application[96] Synthetic molecular mimics of tumor suppressor miR-NAs or the inhibition of oncogenic miRNAs by chemicallymodified antisense oligonucleotides (ASOs) have beenwidelytested in preclinical trials Chemical modifications includ-ing 21015840-O-methyl 21015840-O-methoxyethyl 21015840-fluoro and lockednucleic acid (LNA) have improved the stability biodistribu-tion and delivery of ASOs We have performed preclinicalevaluation of systemic LNA treatment for osteosarcomastem cells Among three upregulated miRNAs in the SaOS2CD133high population miR-133a regulated cell invasion andthe upregulated levels were significantly correlated withpoor prognosis of patients [77] Inversely the silencing ofmiR-133a with LNA reduced cell invasion and the systemicadministration of LNA along with cisplatin suppressed lungmetastasis The tumor expression levels of miR-133a werereduced by LNA administration without a drug deliverysystem Currently a multicenter phase I study involving theliposomal injection of MRX34 an miR-34 mimic for 5 dayswith 2 weeks off is in progress for patients aged over 18 yearswith unresectable primary liver cancer advanced metastaticcancer with or without liver metastasis (melanoma lungcancer) lymphoma and multiple myeloma LNA has alsoundergone a clinical phase II trial as miravirsen (SPC3649)for chronic hepatitis C (HCV) Miravirsen works mainly by

hybridizing with mature miR-122 a liver-specific miRNAwith an important role in the life cycle of HCV and alsobinds to the stem-loop structure of pri- and pre-miR-122 [97]To date this compound has not been tested for malignantdiseases [92]

55 Salinomycin Salinomycin is a polyether ionophoreantibiotic which has been widely used as an anticoccidiosisagent in chickens [87] It was identified in a chemical screenthat had been designed to discover compounds with selectivetoxicity for breast CSCs [98] Tang et al identified thatsalinomycin inhibited osteosarcoma by selectively targetingCSCs both in vitro and in vivo without severe side effects[99] They further identified that downregulation of theWnt120573-catenin self-renewal pathway might contribute to theinhibitory effects of salinomycin on osteosarcoma stem cells[99] However salinomycin displays poor aqueous solubil-ity that hinders its clinical application [89] Thus Ni etal developed salinomycin-loaded PEGylated polynanopar-ticles conjugated with CD133 aptamers (Ap-SAL-NP) andevaluated their cytotoxicity to CD133+ cells Ap-SAL-NPexhibited specific cytotoxicity toward SaOS2 CD133+ cellsand intravenous injections via the tail vein of tumor-bearingmice exhibited significant antitumor activity compared withsalinomycin and a control compound [89] However theredoes not seem to be any information on clinical trials of thesecompounds

56 Bufalin Bufalin is the active ingredient of the Chinesemedicine Chan Su which is extracted from dried toadvenom from the skin glands of Bufo gargarizans [100]This compound inhibits the proliferation of hepatocellularcarcinoma and induces apoptosis in cancer cell lines ofleukemia prostate cancer gastric cancer and osteosarcoma[100] Chang et al identified that bufalin induced the shrink-age of tumor spheres via the activation of caspase-3 anddownregulated stem cell markers including ALDH1 TERTNanog CD133 Notch and Bmi1 miR-148 was found to bea target of bufalin which regulates DNMT1 and p27 [100]A clinical trial of huachansu which includes bufalin withgemcitabine for pancreatic cancer has been completed butthe results are not currently available [92]

6 Conclusions and Perspectives

Substantial effort has been expended on the identification andcharacterization of bone sarcoma stem cells To date variousCSCmarkers have been identified especially in osteosarcomawhichmight reflect the histological and genetic heterogeneityof this tumor Thus a definitive marker for osteosarcomastem cells remains to be found and there seems to be hetero-geneity even within the isolated CSC fraction Furthermoreonly conventional CSC markers have been analyzed forchondrosarcoma Therefore we have to conclude that CSCresearch in bone sarcomas is still at an immature stage andfurther research is required to fully understand the CSCmarkers and their functions In addition no investigationshave been performed on the correlation between CSC mark-ers and the drug-resistant fractions within clinically resected


specimens after neoadjuvant chemotherapy or metastaticspecimens at the early phase of tumor formation Furtherinvestigation using a number of clinically resected specimensand elucidation of the clinical relevance will be important forthe precise characterization of bone sarcoma stem cells

To date most reports have focused on the common CSCmarkers such as CD133 or ALDH which would be reasonableif the sarcoma stem cell subpopulations emerge after theaccumulation of further epigenetic or genetic alterations in asubset of tumor cells However if sarcomas are derived froma single cell that is transformed into a sarcoma stem cellthe mesenchymal stem cell markers such as CD117Stro-1 orCD271 and a neural crest cell marker such as CD57 wouldbe reasonable for sarcomagenesis Nevertheless the positivityrate of these mesenchymal stem cell markers in the clinicalspecimens was negative to quite low [25] indicating thatfurther investigation is necessary to confirm their potentialas bone stem cell markers Despite the inconsistency in themarkers of bone sarcoma stem cells clinical problems thatmust be overcome are drug resistance and metastasis Themolecular regulations that modulate these phenotypes withclinicopathological relevance such as Wnt120573-catenin andmiRNAsmight be important Some of the tumor-suppressivemiRNAs directly regulate the oncogenic fusion gene EWS-FLI-1 and indirectly target CSC markers (Figure 1) therebyanticipating their clinical application asmiRNA therapeuticsClinical benefit will be provided by anti-CSC compounds thatpossess a broad spectrum to the lethal phenotypes and are ofprognostic significance

Among the anti-CSC compounds tested in the reportedstudies some have already undergone clinical trials for othermalignant diseases which will also be promising for sarcomapatients The anti-CSC compounds under clinical trials forother malignancies but not for bone sarcomas include ROR1inhibitor oncolytic adenovirus and immunotherapy [87]Receptor tyrosine kinase-like orphan receptor 1 (ROR1) atyrosine kinase-like cell surface protein that is expressed dur-ing embryogenesis is associated with Ewing sarcoma as wellas breast and ovarian cancer CSCs [101 102] Cirmtuzumabwhich binds with high affinity to ROR1 is currently beinginvestigated in patients with chronic lymphocytic leukemiawho are ineligible for chemotherapy [87 102] A telomerase-specific oncolytic adenovirus (OBP-301) has already beenreported to be effective for the treatment of bone andsoft-tissue sarcomas [103] Indeed this oncolytic virus waspreviously confirmed to be effective by targeting gastricCSCs [104] Kano et al found that the SP cells showedthe expression of human leukocyte antigen (HLA) class Imolecules on the cell surface and the CTL clone Tc4C-6 induced by mixed lymphocyte tumor cell culture usingautologous peripheral blood mononuclear cells and freshlyisolated SP cells showed specific cytotoxicity against theSP cells [105] These compounds could be candidates toovercome bone sarcoma stem cells but have not yet beentested preclinically for bone sarcomas

Current clinical trials will clarify the efficacy of the anti-CSC compounds in the near future Since these compoundsare effective at least preclinically their drug screening in vivoshould clarify which are the most effective and the least toxic

However a difficult issue is how to confirm that these anti-CSC compounds are clinically effective for CSCs Indeedthese compounds have been clinically applied in combinationwith current chemotherapeutic drugs If a favorable outcomewere observed the trial would be a clinical success Thenresearchers would have to carefully investigate the effect onCSCs using clinical specimens to confirm the efficacy of anti-CSC compounds At this stage the inconsistency of CSCmarkers is a major problem and the development of a novelbiomarker for detecting CSCs is required Liquid biopsytechnologies including circulating tumor cells circulatingcell-free DNA circulating cell-free miRNA and circulat-ing exosomes might solve these problems as noninvasivebiomarkers [106] which have not yet been analyzed usingclinical samples from patients with bone sarcomas

Since bone sarcoma is a rare malignant disease globalcooperation among basic researchers and clinicians will berequired to improve its prognosis Although CSC studieson bone sarcoma are in their infancy compared with stud-ies on other malignancies the development of anti-CSCcompounds is highly anticipated These trials should yieldnovel treatment strategies for bone sarcoma patients againsta background in which the clinical outcomes have almostplateaued for 10 years

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported in part by the Grant-in-Aid of theJapan Society for the Promotion of Science (JSPS) KAKENHIGrant nos 16H05449 and 16K20054 and Japan Orthopaedicsand Traumatology Research Foundation Inc no 311

References

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[2] HDDorfman and B Czerniak ldquoBone cancersrdquoCancer vol 75no 1 pp 203ndash210 1995

[3] G Bacci A Longhi M Versari M Mercuri A Briccoli andP Picci ldquoPrognostic factors for osteosarcoma of the extremitytreated with neoadjuvant chemotherapy 15-year experience in789 patients treated at a single institutionrdquo Cancer vol 106 no5 pp 1154ndash1161 2006

[4] S S Bielack B Kempf-Bielack G Delling et al ldquoPrognosticfactors in high-grade osteosarcoma of the extremities or trunkan analysis of 1702 patients treated on neoadjuvant cooperativeosteosarcoma study group protocolsrdquo Journal of Clinical Oncol-ogy vol 20 no 3 pp 776ndash790 2002

[5] P A Meyers G Heller J Healey et al ldquoChemotherapy for non-metastatic osteogenic sarcoma the memorial sloan-ketteringexperiencerdquo Journal of Clinical Oncology vol 10 no 1 pp 5ndash151992


[6] W S Ferguson and A M Goorin ldquoCurrent treatment ofosteosarcomardquo Cancer Investigation vol 19 no 3 pp 292ndash3152001

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[8] V Subbiah P Anderson A J Lazar E Burdett K Raymondand J A Ludwig ldquoEwingrsquos sarcoma standard and experimentaltreatment optionsrdquoCurrent Treatment Options in Oncology vol10 no 1-2 pp 126ndash140 2009

[9] S P F T Nota Y Braun J H Schwab C N Van Dijkand J A M Bramer ldquoThe identification of prognostic factorsand survival statistics of conventional central chondrosarcomardquoSarcoma vol 2015 Article ID 623746 11 pages 2015

[10] A Halldorsson S Brooks S Montgomery and S GrahamldquoLungmetastasis 21 years after initial diagnosis of osteosarcomaa case reportrdquo Journal of Medical Case Reports vol 3 article9298 2009

[11] H Clevers ldquoThe cancer stem cell premises promises andchallengesrdquo Nature Medicine vol 17 no 3 pp 313ndash319 2011

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[13] J E Visvader and G J Lindeman ldquoCancer stem cells in solidtumours accumulating evidence and unresolved questionsrdquoNature Reviews Cancer vol 8 no 10 pp 755ndash768 2008

[14] J E Visvader ldquoCells of origin in cancerrdquo Nature vol 469 no7330 pp 314ndash322 2011

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[85] L Yu S Liu C Zhang et al ldquoEnrichment of human osteosar-coma stem cells based on hTERT transcriptional activityrdquoOncotarget vol 4 no 12 pp 2326ndash2338 2013

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of NF-120581B signaling cascade in MDR-induced stem like cancer-initiating cellsrdquo International Journal of Oncology vol 46 no 6pp 2573ndash2585 2015

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stem cell-like phenotype by histone deacetylase inhibitors insquamous cell carcinoma of the head and neckrdquoCancer Sciencevol 104 no 11 pp 1468ndash1475 2013

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[98] P BGupta T TOnderG Jiang et al ldquoIdentification of selectiveinhibitors of cancer stem cells by high-throughput screeningrdquoCell vol 138 no 4 pp 645ndash659 2009

[99] Q-L Tang Z-Q Zhao J-C Li et al ldquoSalinomycin inhibitsosteosarcoma by targeting its tumor stem cellsrdquo Cancer Lettersvol 311 no 1 pp 113ndash121 2011

[100] Y Chang Y ZhaoW Gu et al ldquoBufalin inhibits the differentia-tion and proliferation of cancer stem cells derived from primaryosteosarcoma cells through Mir-148ardquo Cellular Physiology andBiochemistry vol 36 no 3 pp 1186ndash1196 2015

[101] B Cui S Zhang L Chen et al ldquoTargeting ROR1 inhibitsepithelial-mesenchymal transition and metastasisrdquo CancerResearch vol 73 no 12 pp 3649ndash3660 2013

[102] S Zhang B Cui H Lai et al ldquoOvarian cancer stem cells expressROR1 which can be targeted for anti-cancer-stem-cell therapyrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 111 no 48 pp 17266ndash17271 2014

[103] T Sasaki H Tazawa J Hasei et al ldquoPreclinical evaluation oftelomerase-specific oncolytic virotherapy for human bone andsoft tissue sarcomasrdquo Clinical Cancer Research vol 17 no 7 pp1828ndash1838 2011

[104] S Yano H Tazawa Y Hashimoto et al ldquoA genetically engi-neered oncolytic adenovirus decoys and lethally traps quiescentcancer stemndashlike cells in SG

2M phasesrdquo Clinical Cancer

Research vol 19 no 23 pp 6495ndash6505 2013[105] M Kano T Tsukahara M Emori et al ldquoAutologous CTL

response against cancer stem-like cellscancer-initiating cells ofbone malignant fibrous histiocytomardquo Cancer Science vol 102no 8 pp 1443ndash1447 2011

[106] NKrawczyk FMeier-StiegenM BanysHNeubauer E Ruck-haeberle and T Fehm ldquoExpression of stem cell and epithelial-mesenchymal transition markers in circulating tumor cellsof breast cancer patientsrdquo BioMed Research International vol2014 Article ID 415721 11 pages 2014

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


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International Journal of

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Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


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Stem CellsInternational



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gene and amember of the ETS family of transcription factorsmost commonly FLI-1 [7] Although multidisciplinary careincorporating advances in diagnosis surgery chemotherapyand radiation has substantially improved the survival rateof patients with localized Ewing sarcoma to nearly 70 [8]survival in a metastatic or recurrent disease setting remainsextremely low at lt20 Chondrosarcoma a malignantgroup of cartilaginous matrix-producing neoplasms typicallyoccurring in the fifth to seventh decades of life is generallyresistant to chemotherapy and radiotherapy while Ewing sar-coma is relatively sensitive [1] Its prognosis depends largelyon the histological grade and treatment is mostly limited tosurgical resection [9]

The clinical outcomes of these bone sarcomas haveplateaued for the last 10 years Considering the characteristicsand heterogeneity of bone sarcomas it is possible that asubset of tumor cellsmight resist various stresses and producerecurrence ormetastasis which corresponds to the hallmarksof cancer stem-like cells (CSCs) Indeed there are no fewerbone sarcoma cases involving metastases long after initialtreatments [10] Although targeted therapy for bone sarcomastem cells has not been available several preclinical trials havebeen reported which might improve patient prognosis Thispaper provides an overview of the accumulating knowledgeon bone sarcoma stem cells and preclinical analyses toovercome their lethal phenotypes

2 Cancer Stem Cell Hypothesis inBone Sarcomas

The cancer stem cell hypothesis is based on the observationthat not all cells in tumors are equal [11] It proposes thatthere is a small subpopulation of cells within a heterogeneoustumor that are responsible for forming the bulk of the tumor[12 13] These cells are similar to normal stem cells and mayarise from the transformation of stem cells or the dedifferen-tiation of nonstem cells [14] The common consensus is thatthey are able to self-renew and differentiate into all of thecells within a tumor [12] The first evidence of the existenceof CSCs was reported in hematological malignancies [11]with these cells being characterized as the CD34+CD38minusfraction [15] CSCs have now been isolated from varioushuman solid tumors including bone sarcomas [13] The firstdemonstration of the existence of bone sarcoma stem cellswas achieved by Gibbs et al in 2005 who showed thatosteosarcoma and chondrosarcoma cells include a subpopu-lation of cells that are capable of growing in spheres and havethe properties of self-renewability and multipotency [16]Thereafter several CSC markers that are common to othermalignant diseases as well as unique to bone sarcomas havebeen identified (Figure 1) Recent investigation has focusedon themolecularmechanisms underlying bone sarcoma stemcells and therapeutic testing using preclinical models

3 Characterization of Bone SarcomaStem Cells

Since the first identification of osteosarcoma stem cells byGibbs et al in 2015 various reports on osteosarcoma and

Ewing sarcoma stem cell have been reported while beinglimited for chondrosarcoma Most of these reports havedocumented the conventional CSC markers such as CD133ALDH and side population Notably the mesenchymalstem cell markers such as CD117Stro-1 and CD273 havebeen included which are the unique characteristic of bonesarcoma stem cells (Figure 1 Table 1) However there is noclear consensus on the definite marker that characterizesbone sarcoma stem cells and discussions of several markerssuch as side population have caused controversies in theliterature

31 Sarcosphere (Osteosarcoma Ewing Sarcoma and Chon-drosarcoma) Gibbs et al first reported that several osteosar-coma cell lines established from biopsy specimens andMG63OS cell line formed spheres at a frequency of 01ndash1 in ananchorage-independent environment which could form sec-ondary spheres after the dissociation of single cells [16]Wanget al also identified sphere formation from OS99-1 MG63HuO9 and SaOS2 [17] and Wilson et al found similaritiesbetween human and canine osteosarcoma cell lines in termsof forming spheres [18] Fujii et al further identified thatMG63 spheres showed resistance to doxorubicin and cisplatinand increased expression of the DNA repair enzyme genesMLH1 andMSH2 indicating that a DNA repair inhibitor hadthe potential to enhance the efficacy of chemotherapy [19]

Human Ewing sarcoma cell line also formed spheres at alow frequency rate which expressed higher levels of Oct34Nanog STAT3 Sox2 Sox10 and EWS-FLI1 and showedhigher drug resistance similarly to OS cells [19] Similarlychondrosarcoma cells that originated from patient biopsiesformed spherical colonies which displayed Stro-1 CD44 andCD105 the mesenchymal stem cell markers [16] HoweverLeuchte et al demonstrated that sphere formation is nota reliable method to enrich CSCs of Ewing sarcoma sincethese spheres did not continuously self-renew by secondarysphere formation and sphere culture did not enhance thetumorigenicity in vivo [20] Therefore the sphere modelmight be inadequate to reflect CSC properties but at leastuseful for preclinical in vitro testing of novel therapies

32 CD133 (Osteosarcoma Ewing Sarcoma and Chondrosar-coma) CD133 first recognized as a novel antigen on CD34+progenitor hematopoietic stem cells is a glycoprotein with afive-transmembrane-domain protein encoded by the PROM1gene [21ndash23] Tirino et al were the first to demonstrate thatCD133+ osteosarcoma cells possessed CSC phenotypes Theyshowed that SaOS2 MG-63 and U2OS cell lines containeda small fraction of CD133+ cells ranging from 3 to 5which showed the following phenotypes high proliferationrate cell cycle detection in G

2M phase positivity for Ki-

67 formation of spheres and inclusion of a small subset ofSP cells (097) [24] They further identified that all humanosteosarcoma and chondrosarcoma samples contained asmall population ofCD133+ cells [25]Moreover CD133+ cellsof two stabilized cell lines from clinical samples (50ndash78)also showed self-renewability sphere formation adipogenicand osteogenic differentiation high expression of stemnessgenes and tumorigenicity [25]


Enoxacinb

MST312a BRM270a

HIF2PUTa

TSSC3a Bufalina

Salinomycina

TRBP2b


miR-1247a

LNAa

miR-133aa

EWS-FLI-1

BMP2a

Wnta

Notcha

CD57bCD271a

Sox2aOct34abc

CD47a

ALDHab

CD248a

Stro-1aCD117c-kita

CD133abcd

Telomerasea MC1742a

YK-4-209b

NF-120581Ba

TGF-120573a

TNF-120572a










Osteosarcoma




[24ndash26]





[25 41]









Ewing sarcoma



[19 20]



tumorigenicity

[29 30]































































Osteosarcoma


0G1


PARP

[87]






[95]



[77]




[89]



targeting miR-148

[100]

Ewing sarcoma




[81]

























Competing Interests


Acknowledgments


References
























































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Enoxacinb

MST312a BRM270a

HIF2PUTa

TSSC3a Bufalina

Salinomycina

TRBP2b


miR-1247a

LNAa

miR-133aa

EWS-FLI-1

BMP2a

Wnta

Notcha

CD57bCD271a

Sox2aOct34abc

CD47a

ALDHab

CD248a

Stro-1aCD117c-kita

CD133abcd

Telomerasea MC1742a

YK-4-209b

NF-120581Ba

TGF-120573a

TNF-120572a










Osteosarcoma




[24ndash26]





[25 41]









Ewing sarcoma



[19 20]



tumorigenicity

[29 30]































































Osteosarcoma


0G1


PARP

[87]






[95]



[77]




[89]



targeting miR-148

[100]

Ewing sarcoma




[81]

























Competing Interests


Acknowledgments


References
























































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Osteosarcoma




[24ndash26]





[25 41]









Ewing sarcoma



[19 20]



tumorigenicity

[29 30]































































Osteosarcoma


0G1


PARP

[87]






[95]



[77]




[89]



targeting miR-148

[100]

Ewing sarcoma




[81]

























Competing Interests


Acknowledgments


References
























































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















































Osteosarcoma


0G1


PARP

[87]






[95]



[77]




[89]



targeting miR-148

[100]

Ewing sarcoma




[81]

























Competing Interests


Acknowledgments


References
























































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









































Osteosarcoma


0G1


PARP

[87]






[95]



[77]




[89]



targeting miR-148

[100]

Ewing sarcoma




[81]

























Competing Interests


Acknowledgments


References
























































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















Osteosarcoma


0G1


PARP

[87]






[95]



[77]




[89]



targeting miR-148

[100]

Ewing sarcoma




[81]

























Competing Interests


Acknowledgments


References
























































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Osteosarcoma


0G1


PARP

[87]






[95]



[77]




[89]



targeting miR-148

[100]

Ewing sarcoma




[81]

























Competing Interests


Acknowledgments


References
























































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















Competing Interests


Acknowledgments


References
























































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Competing Interests


Acknowledgments


References
























































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Review Article Overcoming Therapeutic Resistance of Bone ... · Overview of the Molecular Mechanisms and Therapeutic Targets for Bone Sarcoma Stem Cells TomohiroFujiwara 1,2 andToshifumiOzaki