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to the development of Ewing's sarcoma in the mouse [8]. Importantly, human MSCs are also permissive to EWS-FLI1-mediated Ewing's sar- coma transformation.
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Epigenetic and epigenomic mechanisms shape sarcoma and other mesenchymal tumor pathogenesis Sarcomas comprise a large number of rare, histogenetically heterogeneous, mesenchymal tumors. Cancers such as Ewing’s sarcoma, liposarcoma, rhabdomyosarcoma and synovial sarcoma can be generated by the transduction of mesenchymal stem cell progenitors with sarcoma-pathognomonic oncogenic fusions, a neoplastic transformation process accompanied by profound locus-specific and pangenomic epigenetic alterations. The epigenetic activities of histone-modifying and chromatin-remodeling enzymes such as SUV39H1/KMT1A, EZH2/KMT6A and BMI1 are central to epigenetic-regulated transformation, a property we coin oncoepigenic. Sarcoma-specific oncoepigenic aberrations modulate critical signaling pathways that control cell growth and differentiation including several miRNAs, Wnt, PI3K/AKT, Sav‑RASSF1‑Hpo and regulators of the G1 and G2/M checkpoints of the cell cycle. Herein an overview of the current knowledge of this rapidly evolving field that will undoubtedly uncover additional oncoepigenic mechanisms and yield druggable targets in the near future is discussed. KEYWORDS: epigenetic modifiers n fusion oncogenes n histone demethylase n histone marks n histone methyltransferase n mesenchymal tumors n Polycomb group proteins n sarcoma n stem cells

Of mesenchymal stem cells & sarcoma in mice & men Akin to George Milton and Lennie Small, the two tragic characters in John Steinbeck’s novel (Of Mice and Men) [1] , sarcoma and mesenchymal stem cells (MSCs) appear to walk hand-in-hand in the treacherous roads of multipotency, differentiation, and transformation. In the past few years, seminal work from the Stamenkovic group at the University of Lausanne, Switzerland and from others has provided clear evidence supporting MSCs as the cells of origin of sarcomas [2–12] . Thus, for instance, transduction of EWS‑FLI1 Ewing’s sarcoma fusion oncoprotein into primary bone marrow-derived murine MSCs leads to the development of Ewing’s sarcoma in the mouse [8] . Importantly, human MSCs are also permissive to EWS‑FLI1-mediated Ewing’s sarcoma transformation. This imparts a characteristic Ewing’s sarcoma transcription program that includes the upregulation of EZH2 [10] , an epigenetic stemness factor and major epigenetic determinant of Ewing’s sarcoma pathogenesis [2,7] . Human embryonic neural crest stem cells (NESCs) and derived neuro-MSCs are also readily transformed into Ewing’s sarcoma by EWS‑FLI1, a neoplastic process also accompanied by the upregulation of EZH2 [13] . CDK1dependent phosphorylation of EZH2 in human MSCs suppresses its epigenetic activity and leads to osteogenic differentiation, indicating

that EZH2 may also play a role in osteosarcoma pathogenesis [12] . Similarly to the effect of EWS‑FLI1 on MSCs, transduction of the liposarcoma-specific TLS-CHOP (also known as FUS-DDIT3) into MSCs gives rise to myxoid liposarcoma, and MSC transduction with the synovial sarcoma-specific SS18‑SSX1 leads to synovial sarcoma [3,9] . Additional substantiation of the MSC origin of sarcoma includes evidence that deregulation of Wnt signaling in MSCs leads to malignant fibrous histiocytoma (also known as undifferentiated pleomorphic sarcoma not otherwise specified; WHO, 2002 classification [14]), one the most frequent sarcoma of bone and soft tissues in adults [4] . Moreover, both PAX3‑FKHR and PAX7-FKHR alveolar rhabdomyosarcoma oncofusions cooperate with p53 inactivation and activated Ras to produce alveolar rhabdomyosarcoma in murine MSCs [6] . Further evidence of the MSC origin of sarcoma stems from reports of spontaneous transformation of MSCs into sarcoma in vitro and in the mouse [4,10] , as well as in patients recipient of allogeneic or autologous MSC transplantation [15–17] , prompting a cautionary note on the use of MSCs in human therapies [16,18] .

10.2217/EPI.11.93 © 2011 Future Medicine Ltd

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Idriss M Bennani-Baiti Children’s Cancer Research Institute, Vienna, Austria Tel.: +43 140 470 4045 Fax: +43 140 470 7150 [email protected]

MSC epigenomic landscape, a major determinant of sarcomagenesis It is becoming increasingly evident that an inherent differentiation and transformation ISSN 1750-1911

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plasticity makes MSCs uniquely positioned to generate so many different tissues and tumors (Boxes 1 & 2) . Available data indicate that this plasticity is at least in part epigenetically encoded. Thus, for instance, transduction of SS18‑SSX1 induces a transcriptional program in MSCs that includes imprinted genes and chromatinrelated genes [3] . Analysis of several human MSC batches prior to transformation shows differences in genomic imprinting at the IGF2 locus, accompanied with either monoallelic or biallelic IGF2 expression. The difference in the baseline epigenetic state between MSC batches could explain the difference in human MSC permissiveness to synovial sarcoma tumorigenesis, providing a possible molecular basis to the low frequency of synovial sarcoma [3] . Similarly, analysis of ten different imprinted genes on chromosomes 6, 7, 11 and 15 implicates changes in genomic imprinting in the transformation of a model system of human osteosarcoma [19] . Furthermore, stepwise in vitro transformation of MSCs was shown to induce a gradual loss of genomic methylation, indicating that tumorigenesis can cause epigenomic alterations during sarcomagenesis [20] . These and other considerations (see below) indicate that a better understanding of MSC and sarcoma epigenomics may yield important insights into the mechanisms of sarcomagenesis. In the following sections, the most well-documented genes whose epigenetic activation or inactivation can contribute to mesenchymal tumor pathology is summarized (Figure 1) .

Epigenetic alterations at specific gene loci are crucial to the pathology of select mesenchymal tumors The research groups of Stephen Henderson at University College London, UK, and Paul Meltzer at the NCI of the NIH were the first to conduct genomic analyses of large panels of different mesen­chymal tumors. These studies laid the foundations of comparative mesenchymal tumor genomics, and uncovered both shared Box 1. Examples of mesenchymal-derived tissues and cancer counterparts. ƒƒ The mesenchyme is constituted of undifferentiated cells that arise during embryogenesis mostly from the mesoderm. Mesenchymal stem cells give rise to myoblasts, which can differentiate into skeletal muscle (corresponding tumors are embryonal and alveolar rhabdomyosarcoma) or smooth muscle (tumors: leiomyosarcoma), stromal cells (normal: fibrous tissue; tumor: malignant fibrous histiocytoma), osteoblasts (normal: bone; tumor: osteosarcoma), chondroblasts (normal: cartilage; tumor: chondrosarcoma) and adipoblasts (normal: fat; tumor: liposarcoma)

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and distinct expression determinants that characterize the various mesenchymal tumors [21,22] . A large body of work has since been devoted to uncovering the epigenetic bases of gene regulation in these neoplasms, and we survey below those that impact mesenchymal tumor cell growth or differentiation. „„ APC APC is a tumor suppressor that is best known for its antagonistic activities of Wnt signaling. Upon Wnt signaling activation, the APC/Axin/GSK3b complex is inactivated by dishevelled and no longer phosphorylates b‑catenin. The latter translocates into the nucleus and together with TCF4 (also known as TCF7L2 [23]), activates Wnt target genes such as MYC and cyclin D1 to promote mitogenesis. Mutations in APC can cause autosomal dominant premalignant lesions called familial adenomatous polyposis. While loss of heterozygosity analyses and sequencing of the APC locus did not uncover (genetic) mutations in liposarcoma, epigenetic analyses showed that 45% of myxoid/ round-cell liposarcoma were CpG‑methylated at the APC locus and had reduced APC expression [24] . Any effect that this may have on liposarcoma pathology does not appear to be Wntmediated, since b‑catenin protein levels were unaltered in the tumors harboring epigenetically inactivated APC [24] . APC, however, can modulate DNA repair and replication, cell division, differentiation and migration, as well as other cellular processes in a Wnt-independent manner [25] , suggesting that APC inactivation may contribute to liposarcoma pathology through Wnt-independent mechanisms. While there currently are no other published reports on APC epigenetic alterations in sarcomas other then liposarcoma, a recent report of truncating inactivating APC mutations in Ewing’s sarcoma and leiomyosarcoma cell lines suggests that APC inactivation may play an important role in several different sarcomas [26] . „„ Ezrin Ezrin is a member of the ezrin-radixin-moesin family of genes that organizes membrane and cytoskeleton-associated complexes by serving as a bridge between the plasma membrane and the actin cytoskeleton. Ezrin is upregulated in metastases of breast cancer, melanoma, pancreatic adenocarcinoma and prostate cancer [27] . In sarcomas, Ezrin was shown to be crucial to osteosarcoma [28,29] and rhabdomyosarcoma metastasis [30] . In a murine rhabdomyosarcoma future science group

Epigenetic & epigenomic mechanisms shape sarcoma pathogenesis

cell model, Ezrin shows signs of epigenetic activation including CpG‑hypomethylation and H3K9‑acetylation in highly metastatic cells, and conversely CpG‑hypermethylation and low H3K9‑acetylation levels in poorly metastatic cells [27] . Treatment of the latter with the DNA methylation inhibitor 5‑aza-2´-deoxcytidine (decitabine or 5‑aza) induces Ezrin DNA demethylation, Ezrin upregulation and enhances the metastatic potential of these cells. Similarly, treatment with the histone deacetylase inhibitor (HDACi) trichostatin A increases Ezrin H3K9‑acetylation and expression levels, and potentiates rhabdomyosarcoma metastasis. Importantly, an Ezrin-targeting shRNA blocks both 5‑aza- and trichostatin A-induced rhabdomyosarcoma metastasis, indicating that epigenetic activation of Ezrin in these cells is responsible for rhabdomyosarcoma metastasis [27] . Whilst Ezrin epigenetics has not been explored in other sarcomas, high Ezrin expression levels correlate with higher tumor grade in osteosarcoma and chondrosarcoma [31] , and drives Ewing’s sarcoma cell growth [32] , suggesting a role for Ezrin in the aggressive behavior of several different sarcomas. „„ FGFR1 FGFR1 overexpression, amplification, or associated chromosomal translocations were uncovered in several different cancers, pointing to FGFR1 as a bona fide proto-oncogene [33] . FGFR1 is a tyrosine kinase receptor and a potent mitogen that suppresses myoblast differentiation and promotes myogenic cell proliferation. It is preferentially expressed in the developing mesenchyme and is thought to facilitate epithelial-mesenchymal transition as well as myoblast neoplastic transformation [34–36] . Evidence of epigenetic activation of FGFR1 in rhabdomyosarcoma was revealed in a comparative analysis of the FGFR1 locus in rhabdomyosarcoma and normal skeletal muscle, whereby an FGFR1 5´ CpG island was found to be hypomethylated in rhabdomyosarcoma, accompanied with FGFR1 overexpression [33] . AKT1, a serine/threonine protein kinase downstream of FGFR1 signaling was also upregulated, suggesting active FGFR1 signaling [33] . There currently are no other reports of FGFR1 epigenetic activation in other sarcomas. It is, however, activated in Ewing’s sarcoma primary tumors and supports Ewing’s sarcoma cell motility and invasiveness [37] , showing that FGFR1 plays a role in the pathogenesis of multiple sarcomas through genetic and epigenetic activating mechanisms. future science group

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Box 2. Mesenchymal tumors are rare but varied; some are aggressive and display a predilection for metastasis. ƒƒ Mesenchymal tumors regroup a large number of histogenetically diverse low-incidence neoplasms with approximately 15,000 newly diagnosed sarcomas of soft tissues and bone in the USA per year. High local aggressiveness and metastatic propensity combined with a lack of targeted therapies associates several mesenchymal tumors to high morbidity and mortality. Mesenchymal tumors include alveolar and embryonal rhabdomyosarcoma, chondroblastoma, chordoma, chondromyxoid fibroma, chondrosarcoma, desmoid fibromatosis, dermatofibrosarcoma, Ewing’s sarcoma, fibrosarcoma, gastrointestinal stromal tumor, hemangiopericytoma, leiomyosarcoma, liposarcoma, malignant fibrous histiocytoma, mixed Müllerian tumor, malignant peripheral nerve sheath tumors, synovial sarcoma, neurofibroma, osteosarcoma and schwannoma

„„ GADD45A GADD45A is a genotoxic stress-induced gene downstream of p53, BRCA1 or MAPKdependent signaling. GADD45A regulates biological processes relevant to tumorigenesis including DNA repair and cell cycle by interacting with several proteins including the cyclin-dependent kinase inhibitor p21WAF1/CIP1, proliferating cell nuclear antigen, CDC2 protein kinase, and the MTK/MEKK4 upstream activator of the JNK/SAPK pathway. Induction of GADD45A can lead to cell cycle arrest or to apoptosis, and suppression of GADD45A, therefore, can confer a growth advantage to tumor cells. GADD45A expression is repressed in osteosarcoma cell lines and xenografts via Sarcoma/ locus

EWS

LMS

LPS

MFH MPNST

OST

RMS

SNS

APC CDKN1A CDKN2A FGFR1 GADD45A MGMT MST2/STK3 MST1/STK4 PTEN RASSF1A WIF1

Figure 1. Demonstrated epigenetic alterations in human mesenchymal tumors. Except for the FGFR1 oncogene, the genes listed encode for proteins that are tumor suppressive in sarcomas. Frequent and sporadic methylation aberrations are depicted in dark and light grey, respectively. Arrows pointing up indicate DNA hypermethylation, while those pointing down are synonymous of hypomethylation. The figure gives snapshots of both the multitude of gene loci epigenetically regulated in a specific sarcoma, as well as the variety of sarcomas that may be targeted by epigenetic modulation of a given gene. EWS: Ewing’s sarcoma; LMS: Leiomyosarcoma; LPS: Liposarcoma; MFH: Malignant fibrous histiocytoma; MPNST: Malignant peripheral nerve sheath tumors; OST: Osteosarcoma; RMS: Rhabdomyosarcoma; SNS: Synovial sarcoma.

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mechanisms that involve epigenetic inactivation of GADD45A 5´ upstream and coding sequences [38,39] . Treatment of osteosarcoma cells or of osteosarcoma xenograft-bearing mice with 5‑aza induces GADD45A demethylation and expression, and induces apoptosis in both system models [38] . 5‑aza-induced apoptosis is suppressed by GADD45A shRNA, indicating that GADD45A represents a candidate for epigenetic drug targeting in the treatment of osteosarcoma [38] . „„ MGMT MGMT is a DNA repair enzyme that removes mutagenic and cytotoxic alkyl adducts from guanine residues at O 6 . In the absence of MGMT, O6 -alkylguanine is either converted to adenine during DNA replication thus creating a GàA mutation, or in the case of O6‑chloroethylguanine, crosslinked to the cytosine on the opposite strand, thus blocking DNA replication. One MGMT molecule is inactivated per each DNA lesion repaired, establishing a linear relationship between MGMT cellular levels and the cell’s capacity to withstand mutagenic guanine alkylating agents [40] . MGMT expression is frequently reduced or lost in cancer. A study of sarcomas showed frequent MGMT promoter hypermethylation and loss of expression in liposarcoma and malignant fibrous histiocytoma [41] . Similarly, a study of 30 osteosarcoma primary tumors and metastases showed MGMT to be more methylated in osteosarcoma then in matching normal tissues from the same patients [42] . Furthermore, analysis of 65 mesenchymal tumors showed that MGMT is also hypermethylated in 15–18% of leiomyosarcoma, malignant fibrous histiocytoma and malignant peripheral nerve sheath tumors [43] . Hypermethylated MGMT is ten-times more likely to associate with large-size (≥10 cm) rather than small-size sarcomas, and is 14-times more frequent in advanced III/IV stages compared with early I/II sarcoma stages [43] . Accordingly, loss of MGMT expression in sarcomas correlates to poor survival [41] , underscoring the importance of this enzyme to the biology of sarcoma, and the potential of MGMT-activating drugs in sarcoma therapy. „„ STK3 & STK4 STK3 and STK4 are apoptotic kinases and human homologs of the yeast ‘sterile 20’ and Drosophila melanogaster proapoptotic Hippo (Hpo) kinases. In Drosophila, activation of a Sav‑RASSF1‑Hpo pathway leads to cyclin  E downregulation, cell cycle arrest and apoptosis. 718

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In humans, STK3 is activated by various stress stimuli, including the proapoptotic straurosporine and FAS ligand, whereas STK4 acts upstream of the stress-induced MAPK cascade and phosphorylates histone H2B during apoptosis. Both STK3 and STK4 localize to the microtubules and interact with RASSF1A to regulate apoptosis. Analysis of STK3 methylation showed STK3 to be hypermethylated in 15% of liposarcoma, a third of malignant fibrous histiocytoma and myxofibrosarcoma and a half of synovial sarcoma [44] . Analysis of the STK4 locus showed it to be hypermethylated in about one fifth of malignant fibrous histiocytoma and myxofibrosarcoma, one fourth of liposarcoma, a half of leiomyosarcoma and 80% of rhabdomyosarcoma tested, and was accompanied with reduced STK4 expression [44] . STK4 hypermethylation was confirmed in leiomyosarcoma and rhabdomyosarcoma cell lines, providing in vitro cell models to explore the effects of epigenetic activation of STK4 on the Sav‑RASSF1‑Hpo tumor suppressor pathway and biology of these tumors. The RASS1FA locus was also hypermethylated in approximately a fifth, 40%, and a half of liposarcoma, leiomyosarcoma, and neurogenic sarcoma, respectively [44] . These data indicate that epigenetic inactivation of the Sav‑RASSF1‑Hpo tumor suppressor pathway by CpG DNA hypermethylation is a relatively frequent occurrence that may represent a common epigenetic target in mesenchymal tumors, and opens the door to possible common epigenetic therapies. Epigenetic inactivation of components of the Sav‑RASSF1‑Hpo tumor suppressor pathway is associated with poor prognosis in lymphoblastic leukemia and with aggressive behavior in breast cancer [45,46] , highlighting the importance of this pathway in cancer. Importantly, mice deficient in LATS1, another component of the Sav‑RASSF1‑Hpo tumor suppressor pathway, develop sarcoma [47] , strongly supporting the relevance of this pathway to sarcoma pathogenesis. „„ The cyclin-dependent kinase inhibitors INK4A/ARF (CDKN2A), p15INK4B (CDKN2B) & p21WAF1/CIP1 (CDKN1A) The cyclin-dependent kinase inhibitor INK4a/ARF locus produces alternatively spliced mRNAs which encode two distinct proteins, p14 ARF and p16INK4a. p14 ARF induces cell cycle arrest at G1 and G2/M by inhibiting MDM2 and stabilizing p53. Its relevance to sarcoma was revealed in p14 ARF knockout mice which future science group

Epigenetic & epigenomic mechanisms shape sarcoma pathogenesis

develop sarcomas at an early age. p16INK4a is a potent tumor suppressor inactivated in many cancers which induces G1 arrest by inhibiting CDK4- and CDK6-dependent phosphorylation of the retinoblastoma protein. p15INK4b is also an important regulator of the G1 checkpoint of the cell cycle that inhibits CDK4- and CDK6associated cyclins. The p21WAF1/CIP1 protein binds and inhibits the CDK2- and CDK4-cyclin complexes, thus serving as a major cell cycle G1 checkpoint. It also interacts with proliferating cell nuclear antigen to regulate DNA replication and DNA repair during the S phase. Both p14 ARF and p16INK4a tumor suppressors are not expressed in several osteosarcoma cell lines due to either homozygous deletion or promoter methylation [48] . Both proteins appear to be tumor suppressive in these cell lines as ectopic expression of either leads to cell cycle arrest and a dramatic decrease in the potential of osteosarcoma cells to form colonies in soft agar [48] . Epigenetic inactivation of p14ARF and/or p16INK4a was confirmed in a study of 30 osteosarcoma primary tumors and metastases which showed a distinct association of methylation to osteosarcoma as compared with normal adjoining tissues [42] . Another study found that hypermethylation of p16INK4a promoter accounts for approximately 60% of cases of reduced p16INK4a expression in osteosarcoma, and in concert with retinoblastoma protein expression status, could predict relapse [49] . Epigenetic inactivation of p16INK4a may represent a major event in the pathogenesis or maintenance of sarcomas. This is, for example, the case in liposarcoma, wherein promoter methylation-mediated epigenetic inactivation of p16INK4a is frequently associated with the highly aggressive dedifferentiated liposarcoma subtype, but not with the less aggressive well-differentiated liposarcoma [50] . In alveolar rhabdomyosarcoma, reduced levels or loss of p16INK4a due to CpG‑methylation-mediated epigenetic inactivation cooperate with the PAX3‑FKHR fusion oncoprotein to bypass a senescence-associated growth arrest checkpoint [51] . Also in rhabdomyosarcoma, 13 out of 26 tumors and two out of five cell lines analyzed showed complete methylation of a signal transducer and activator of transcription-responsive element upstream of the p21WAF1/CIP1 promoter and reduced p21WAF1/CIP1 protein levels [52] . Consistent with these findings, induction or forced expression of p21WAF1/CIP1 in embryonal rhabdomyosarcoma cells leads to growth arrest and myogenic differentiation [53] . Sporadic cases of epigenetic inactivation of the INK4a/ARF or p15INK4B loci were also described future science group

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in other mesenchymal tumors such as Ewing’s sarcoma, leiomyosarcoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumors, and synovial sarcoma [43,54–56] . While it remains to be seen whether DNA methylationmediated epigenetic inactivation of any single cyclin-dependent kinase inhibitor is relevant to sarcomagenesis, epigenetic inactivation of at least one cell cycle inhibitor is observed in approximately 60% of osteosarcoma and Ewing’s sarcoma [56] , suggesting an important role of epigenetic inactivation of cell cycle checkpoints as a means to alleviating tumor suppression in these sarcomas. Accordingly, ectopic expression of the Ewing’s sarcoma pathog­nomonic EWS‑FLI1 fusion oncoprotein in human NESCs is associated with an Ewing’s sarcoma transcriptional signature and epigenetic silencing of p16INK4a that requires the PRC1 component BMI1 [13] . Finally, a study of histiocytic sarcoma showed that 80% of analyzed tumors harbored either a hypermethylated p16INK4a and/or p14ARF, and a mouse model hemizygous for PTEN (see below), in which INK4a/ARF were deleted, showed a biphasic pattern of lymphoblastic lymphoma and histiocytic sarcoma, further confirming the importance of p14ARF and p16INK4a to histiocytic sarcoma genesis [57] . „„ PTEN PTEN is a dual phosphatidylinositol‑3,4,5‑trisphosphate 3‑phosphatase and lipid phosphatase which negatively regulates the PI3K‑AKT‑mTOR oncogenic pathway. CpG‑hypermethylation of PTEN has been associated with several cancers including those of the breast, colorectum, endometrium, liver, lung, prostate, skin, stomach, as well as with hematological malignancies and glioma [58] . Cells appear to be highly sensitive to PTEN dosage as even a 20% reduction in PTEN levels induces tumors in mice [59] , suggesting that hypermethylation of one PTEN allele may be sufficient to promote tumorigenesis. PTEN haploinsufficiency is prevalent in human breast and prostate cancers indicating that reduced PTEN dosage can also be sufficient to support human tumorigenesis; reviewed in [60] . In mesenchymal tumors, 6% of malignant fibrous histiocytoma, 8% leiomyosarcoma and 29–38% of malignant peripheral nerve sheath tumors show hypermethylated PTEN [43,61] . PTEN expression can also be epigenetically inactivated by histone deacetylation following binding of the histone deacetylase (HDAC)-containing Mi‑2/NuRD epigenetic repressor complex to PTEN promoter  [62] . Treatment of synovial sarcoma cells www.futuremedicine.com

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with HDACi leads to indirect PTEN induction and PTEN-mediated apoptosis [63] . PTEN expression can be also induced by caffeine, leading to PI3K‑AKT pathway inactivation and a block of osteosarcoma cell proliferation [64] , indicating that PTEN is a bona fide drug target in sarcoma. „„ RASSF1A RASSF1A is a tumor suppressor which regulates Ras-mediated apoptosis, microtubule stability and mitosis via inhibition of the APC/CDC20 complex [65,66] . RASSF1A is located at 3p21.3, a chromosomal region frequently deleted in several carcinomas. RASSF1A is also frequently inactivated by CpG‑methylation in several cancers including those of the colon, kidneys, bladder, breast, liver, lung, pancreas, prostate, ovaries and skin [67–70] . The importance of RASSF1A epigenetics is underscored by the fact that RASSF1A methylation status can serve as a prognostic marker in several of these cancers [68–70] . In sarcomas, a paired analysis of 30 osteosarcomas and adjacent normal tissues showed more than tenfold higher methylation levels of RASSF1A in osteosarcoma than in normal tissues [42] . Similarly, in a test panel of nine genes that included RASSF1A, p16INK4A , MGMT, RARb, DAPK, APC, GSTP1, CDH1 and CDH13, RASSF1A was the most frequently methylated gene, with over 60% of tested rhabdomyosarcoma showing R ASSF1A hypermethylation compared with normal skeletal muscle [71] . To date, hypermethylation of RASSF1A has been reported to be most frequent in Ewing’s sarcoma (68%), and is associated with low RASSF1A expression and a worse prognosis [72] . Additional mesenchymal tumors wherein RASSF1A hypermethylation-mediated epigenetic inactivation may be involved in the pathogenesis of tumor subsets include malignant fibrous histiocytoma (depending on the study, RASSF1A was found to be hypermethylated in 0–31% of tumors analyzed; average: 14.5%), liposarcoma (18–36.4%; average: 24.2%), leiomyosarcoma (12.5–39%; average: 30.8%), malignant peripheral nerve sheath tumors (18–62.5%; average: 35.5%) and synovial sarcoma (33–47.6%; average: 44.4%) [43,55,61,73] . Finally, stage II–III sarcoma patients with unmethylated RASSF1A have better survival odds than those with methylated RASSF1A (median survival of 58 vs 12 months, respectively) [44] , underscoring the importance of this gene’s epigenetics to sarcoma aggressiveness and patient survival, suggesting that demethylating agents may be useful in the treatment 720

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of sarcoma patients bearing a hypermethylated RASSF1A locus. There is some evidence to that effect as treatment of Ewing’s sarcoma, osteosarcoma, rhabdomyosarcoma and synovial sarcoma cell lines with 5‑aza restored RASSF1A gene expression [44,71–74] . In synovial sarcoma, 5‑aza treatment reduced tumor cell growth, not only in vitro, but also in a mouse xenograft model  [73] , further supporting the premise of RASSF1A targeting in sarcoma therapy. „„ WIF1 WIF1 is a secreted Wnt signaling antagonist that inhibits the Wnt pathway by binding secreted Wnt ligands and blocking their interaction to Wnt receptors. Active Wnt signaling leads to transcriptional activation of genes that drive the cell cycle, such as CDC25A, MYC, MYB, cyclins D and E, and oncogenic mutations of several components of the Wnt pathway, including b‑catenin, E‑cadherin, APC, WNT1, AXIN and TCF7L2/TCF4 have been associated with cancer [75] . In addition to activating oncogenic Wnt mutations, epigenetic silencing of secreted Wnt antagonists affecting DKK1, DKK3, SFRP1, SFRP2, SFRP4 and SFRP5 were also uncovered in several cancers [76–80] , and epigenetic silencing of WIF1 per se was reported in breast, gastrointestinal and lung cancers [79,81,82] . A high-throughput analysis identified WIF1 to be also epigenetically silenced in osteosarcoma cell lines, and 5‑aza-induced WIF1 demethylation and re-expression reduced b‑catenin levels, inhibited osteosarcoma cell proliferation, and induced osteoblastic cell differentiation [83] . Consistent with these findings, Wif1 is highly expressed during murine osteoblastic differentiation and is required for osteoblast differentiation in vitro [83] . In mice, loss of Wif1 leads to both spontaneous osteosarcoma and increased susceptibility to radiation-induced osteosarcoma, suggesting that WIF1 inactivation plays a major role in osteosarcomagenesis [83] . Accordingly, WIF1 expression is suppressed in human osteosarcoma primary tumors concomitant with promoter hypermethylation, loss of WIF1 protein, increased b‑catenin levels and high proliferation [83,84] . Moreover, experimental activation of WIF1 suppresses anchorage-independent osteosarcoma cell growth and motility in vitro, and dramatically reduces the number of lung metastases in an orthotopic mouse model of osteosarcoma, further implicating WIF1 and Wnt in osteosarcoma pathology [84] . Earlier comparative mesenchymal tumor genomic studies had uncovered evidence of future science group

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WNT5A-dependent Wnt pathway activation in synovial sarcoma [21] . More recently, it was reported that approximately half of Ewing’s sarcoma, fibrosarcoma, leiomyosarcoma, liposarcoma, osteosarcoma, rhabdomyosarcoma and synovial sarcoma, as well as 65% of sarcoma cell lines show evidence of Wnt activation [26] . In 16 cell lines representative of these sarcomas, frequent epigenetic inactivation of the SFRP1, SFRP2, SFRP4, SFRP5, DKK1 and DKK2 genes was uncovered [26] , suggesting that methylationdependent Wnt antagonist gene inactivation may be a widespread mechanism of sarcomagenesis. Downmodulation of activated Wnt signaling suppresses sarcoma cell proliferation in vitro and sarcoma xenograft growth in mice [26] . Together with other reports of Wnt signaling activation in synovial sarcoma and uterine soft tissue sarcoma [85–87] , these findings indicate that Wnt signaling constitutes a major determinant of sarcoma pathogenesis through both genetic and epigenetic mechanisms. „„ miRNAs miRNAs are RNA polymerase  II-transcribed small (on average 22 nucleotide), noncoding, ssRNAs that effect post-transcriptional gene silencing. This is achieved either via perfect base pairing of the miRNA with the 3´-UTR of an mRNA target leading to its degradation via the 5´‑3´ mRNA decay pathway, or by translation repression following imperfect base pairing with the mRNA target [88] . miRNAs regulate most biological processes including those pertinent to tumor pathology such as apoptosis, cell cycle, cellular differentiation, development, inflammation, invasiveness, metabolism and stress response [89] . Below, miRNAs whose epigenetic regulation may play a role in sarcoma pathogenesis are discussed. miRNAs can act either as oncogenes and/or as tumor suppressor genes, and examples of the latter include miR-34a and miR-34b/c. Both miR-34a (which targets CDK6) and miR-34b/c (which targets CDK6, CREB, E2F3, MYC, NOTCH1 and NOTCH2) can be epigenetically inactivated in cancers [90–94] . A recent study of eight soft-tissue sarcomas and 40 sarcoma cell lines showed miR-34a to be inactivated by CpG‑methylation in 21 out of 40 cell lines and in six out of eight tumors, and miR-34b/c to be hypermethylated in 16 out of 40 cell lines and six out of eight sarcoma [95] . miR-34b/c is also repressed in osteosarcoma, and an analysis of miR-34b/c in 117 osteosarcoma samples showed that lack of miR-34b/c expression may future science group

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only in part be explained by loss of hetero­ zygosity (18% of cases) or homozygous deletions (less than 1% of cases). The same study showed that approximately 55% of tumors harbor hypermethylated miR-34b/c locus, compared with less than 1% of matching normal tissues from the same patients, indicating that epigenetic inactivation is the predominant mechanisms of miR-34b/c repression in osteosarcoma [96] . Forced expression of miR-34a or miR-34b/c induces cell cycle arrest and apoptosis of osteosarcoma cells through modulation of CDK6, E2F3, cyclin E2 and BCL‑2 expression [96] , indicating that epigenetic inactivation of miR-34a and miR-34b/c may play a role in the pathology of osteosarcoma and possibly other sarcomas. Similarly, miR-29b, a determinant of myogenic differentiation, is repressed in rhabdomyosarcoma [97] . Ectopic expression of miR-29b inhibits rhabdomyosarcoma tumor growth both in vitro and in mice, and induces myogenic differentiation [97] , indicating miR-29b as a bona fide tumor suppressor gene in rhabdomyosarcoma. Comparative analysis of rhabdomyosarcoma and adjoining normal skeletal muscle tissue in patients showed the miR-29b locus to be specifically bound by EZH2 in rhabdomyosarcoma, indicating that miR-29b repression may be epigenetically effected by EZH2 catalysis of H3K27me3 [97] . Additional analyses showed that EZH2, H3K27me3 and HDAC 1 associate with miR-29b chromatin only in the repressed state, further suggesting an epigenetic mechanism of miR-29b repression in rhabdomyosarcoma [97] . Finally, a comparison of miRNA expression profiles between osteosarcoma primary tumors and normal human osteoblasts showed that miR-127–3p, miR-154, miR-154*, miR-299–5p, miR-329, miR-337–3p, miR-376a*, miR-376a, miR-376c, miR-377, miR-382, miR-409–3p, miR-409–5p, miR-410, miR-432, miR-493*, miR-495, miR-543, miR-654–5p and miR-758 were all downregulated in osteosarcoma [98] . These miRNAs are peculiar in that they form a gene cluster on 14q32.31, and array comparative genomic hybridization analyses showed this cytoband to be either normal or amplified in most tumors tested, indicating that repression of these miRNAs in osteosarcoma may not be attributed to reduced copy number, and suggesting epigenetic mechanisms that remain to be determined [98] . Several recent studies started to reveal the role of additional miRNAs in sarcoma biology (e.g., miRNA145 and Let‑7a in Ewing’s sarcoma [99,100]), but any mechanisms www.futuremedicine.com

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of epigenetic regulation of these miRNAs in sarcoma await to be discovered. „„ Chondrosarcoma-specific epigenetics In addition to epigenetic aberrations at gene loci shared by more than one sarcoma (Figure 1) , specific sarcoma subtypes also harbor specific epigenetic events that shape their pathologies. Thus, for instance, an epigenetic switch is associated with chondrosarcoma tumorigenesis during mesenchymal-epithelial transitions. These include loss of CpG‑methylation of genes encoding epithelial markers such as maspin (SERPINB5) and 14–3–3d, a phenomenon that can be re-enacted in chondrocytes following treatment with the demethylating agent 5‑aza [101] . Other tumorigenic processes such as chondrosarcoma invasiveness are also tributary to epigenetic modulation. These include DNA methylation-associated epigenetic inactivation of heparan sulfate 3‑O‑sulfotransferase genes, which encode enzymes that shape the composition and distribution of heparan sulfate proteoglycans, proteins at the core of cancer-relevant biological processes such as cell adhesion and migration [102] .

Large-scale & global epigenetic aberrations cooperate with specific sarcoma-specific genetic lesions to promote oncogenesis As described above, epigenetic modulation of discrete gene loci that encompass oncogenes or tumor suppressor genes can impact sarcoma pathology and present epigenetic therapeutic opportunities. The effect of epigenetics on sarcoma can be more profound when, for instance, a pathognomonic oncogenic transcription factor interacts with chromatin remodeling complexes or with epigenetic modifiers, thus recruiting chromatin remodeling or modifying enzymes to large numbers of gene targets. In addition, sarcoma-specific epigenomic signatures can take place if one or several epigenetic modifiers are up- or down-regulated compared with a normal tissue counterpart. Contrary to the more frequent neoplasms such as those of the breast, colon and liver [103–105] , there have been few sarcoma epigenomic initiatives, at least in part due to limited funding for sarcoma research (Box 3) . Below is a summary of recent large-scale and epigenomic sarcoma research, as well as considerations that indicate that genome-wide sarcoma-specific epigenetic events do take place and can be targeted for therapy. 722

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„„ Epigenetic regulation via recruitment of epigenetic modifiers by sarcoma-specific oncogenes Both Ewing’s sarcoma and synovial sarcoma are cytogenetically characterized by chromosomal translocations that lead to de novo expression of fusion oncoproteins. The vast majority of Ewing’s sarcoma and synovial sarcoma express EWS‑FLI1 and SS18‑SSX fusions, respectively. These fusions are central to driving and maintaining the tumorigenic phenotype and knockdown of EWS‑FLI1 or SS18‑SSX inhibits cell growth and/or leads to apoptosis of their respective sarcomas [106,107] . EWS‑FLI1 is a transcription factor that binds to over 2000 chromatin loci via its ETS DNA binding domain contained within FLI1 [108] . EWS‑FLI1 also interacts with BARD1 and with CBP/p300 [109] . Since both BARD1 and CBP/p300 can interact with the EWS portion of the fusion, EWS‑FLI1 may bind DNA via its FLI1 moiety while interacting with BARD1 or CBP/p300 via EWS, thus potentially recruiting these proteins to chromatin. BARD1 is a component of the BRCA1/BARD1 ubiquitin ligase complex responsible for H2AK119 ubiquitylation and transcriptional repression, and CBP/p300 is a histone acetyltransferase that can acetylate H2AK5, H2BK12, H2BK15, H3K14 and H4K8 and activate transcription. It is therefore possible that EWS‑FLI1 effects, at least in part, its transcriptional program via epigenetic mechanisms that involve histone acetylation or ubiquitylation of some of the gene loci it binds. SS18‑SSX fusions, on the other hand, lack a DNA binding domain but can be recruited to DNA either by MLLT10 (also known as AF10) which interacts with SS18, or by LHX4 which interacts with the SSX moiety of the fusion. It can thus be predicted that SS18‑SSX fusions may bind to MLLT10 and/or LHX4 target genes, though to the best of our knowledge, genome-wide chromatin immunoprecipitation (ChIP) analyses are yet to be reported to test this prediction. SS18‑SSX fusions interact with the BRM, BRG1 and INI1 subunits of the SWI/SNF chromatin remodeling machinery, as well as with the p300 histone acetyltransferase and with SIN3A, a component of the HDAC-containing SIN3A corepressor complex, and could thus potentially modulate chromatin post-translational modifications and structure. The domain within SS18 involved in these interactions is also required for SS18‑SSX fusion-MLLT10 interaction, and it is therefore unlikely that SS18‑SSX fusions future science group

Epigenetic & epigenomic mechanisms shape sarcoma pathogenesis

could recruit these chromatin remodeling and modifying activities to MLLT10-regulated genes as hypothesized by others [110] . It is conceivable, however, that SS18‑SSX fusions could recruit these enzymes to LHX4 regulated genes, whereby the SSX moiety would tether the fusion to DNA-bound LHX4, whilst the SS18 moiety recruits the interacting epigenetic enzymes to chromatin. In addition to LHX4, SSX also interacts with the PHC2, BMI1, and RING1 components of the PRC1, suggesting that SS18‑SSX fusions could effect H2AK119 ubiquitylation at bound genomic loci, but this still awaits experimental confirmation. ChIP analysis of SS18‑SSX2 modulated genes showed that SS18‑SSX2 induces H3K4me3 and H4K16ac, leads to CpG‑hypermethylation of the IGF2 imprinting control region, and its binding to chromatin correlates to H3K27me3 [111] . SS18‑SSX2 also binds to EGR1 tumor suppressor gene promoter, recruits EZH2 and induces H3K27me3 of the EGR1 promoter, both in synovial sarcoma cells and in heterologous cells expressing SS18‑SSX2 [112] . In presence of SS18‑SSX2, EGR1 displays both the H3K27me3 repressing and H3K4me3 activating histone marks that are indicative of bivalent epigenetic marks. Such bivalent marks regulate genes central to developmental control in embryonic stem cells [113] , and of tumor suppressor genes that are repressed in cancer stem cells, but are poised for transcriptional activation [114] . EGR1 is not expressed in synovial sarcoma primary tumors and cell lines [112] , suggesting that SS18‑SSX2 may impart cancer stem cell transcriptional programs through EZH2-dependent EGR1 repression both in vitro and in patients. In vitro, HDACi, FK228 depsipeptide (romidepsin), dramatically reduces H3K27 trimethylation and induces expression of EGR1 [112] , and suppresses synovial sarcoma cell growth [115] , pointing to a crosstalk between EZH2 and HDACs at the EGR1 locus, and indicating that epigenetic means to activating EGR1 may potentially be useful to impeding synovial sarcoma cell growth. „„ Determination of sarcoma-specific epigenomes by aberrant sarcoma-specific epigenetic modifier constellations Epigenetic regulation is orchestrated by the combinatorial expression of a large number of DNA and histone modifying enzymes (HMEs) to effect what has been recognized as the histone code [116,117] . Alterations in the expression of an HME future science group

Review

Box 3. Scarce research funding for scarce cancers. ƒƒ The NCI/NIH budget for research on specific cancers mirrors the incidence and toll that these cancers have on society. Thus, breast, prostate, lung and colorectal cancers account for most cancer cases in the USA and attract most of the funding. The research budget of the NCI/NIH in 2009 was a mere US$40.5 million for all pediatric and adult sarcomas combined, funding that was shy of that for kidney cancer research alone, and less than 7% of the budget allotted to breast cancer research [201]

can lead to an altered, nonphysiological histone code, which in turn can facilitate tumor-favorable transcriptional programs [118,119] . Several recent findings strongly support a role of HMEs in the pathology of mesenchymal tumors. PRC1

PRC1 is composed of BMI1, PC2 (also known as CBX4), PHC3 and RING1. PRC1 is recruited to chromatin at gene loci marked for transcriptional repression by H3K27me3, wherein it condenses chromatin and lessens chromatin accessibility to transcriptional activators. Recent immunohistochemical investigations of 22 chondrosarcoma, 130  Ewing’s sarcoma and 32  osteosarcoma, showed that approximately a half of chondrosarcoma and osteosarcoma, and 80% of Ewing’s sarcoma overexpress BMI1 [120,121] , a protein that drives the self-renewal capacity of both normal and cancer stem cells [122–124] . shRNA-mediated BMI1 knockdown inhibits in vitro osteosarcoma cell growth, colony formation on soft agar and cell migration, and reduces both the number and size of tumors in a xenograft mouse model, implicating BMI1 overexpression as a major contributor to osteosarcoma tumorigenic phenotype [121] . Osteosarcoma tumorigenicity is restored both in vitro and in vivo following introduction of a BMI1 wobble mutant that it is impervious to BMI1-targeting shRNA, further confirming the specificity of BMI1 observed effects. In addition, BMI1 knockdown sensitizes osteosarcoma cells to cisplatin-induced apoptosis through downmodulation of the PI3K/AKT pathway, showing that HME overexpression can cause chemoresistance in sarcoma [121] . Two recent studies show that changes in the composition or activity of the PRC1 complex can also be associated with sarcoma pathology. Thus, in osteosarcoma, an exquisite balance between the BMI1, RING1B and PHC3 components of PRC1 controls cell growth in  vitro [125] . Osteosarcoma cell lines express low levels of PHC3 but high-levels of BMI1 and RING1B [125] . Forced expression of PHC3 leads to downregulation of BMI1 and impaired cell proliferation, and conversely, ectopic expression www.futuremedicine.com

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of BMI1 downmodulates PHC3 expression and boosts cell growth [125] . Similarly to the cell lines, osteosarcoma tumors express low levels of PHC3, and patients that express the lowest PHC3 levels have the lowest survival odds [125] . PRC1 was also shown to be important to synovial sarcoma pathology, wherein SS18‑SSX2 associates with BMI1 and causes destabilization of the Polycomb complex, which leads to derepression of PRC1 gene targets [126] . These include members of the Notch, epherin and platelet derived growth factor pathways, which appear to be oncogenic in synovial sarcoma [126] . In Ewing’s sarcoma, overexpression of BMI1 in patient samples associates with the upregulation of oncogenic IGF1, mTOR and Wnt canonical pathways [120] , suggesting that BMI1 is also involved in Ewing’s sarcoma pathogenesis. Accordingly, BMI1 is induced as part of an Ewing’s sarcoma transcriptional program following forced expression of EWS‑FLI1 in human NESCs [13] . EWS‑FLI1 expression in NESCs bypasses cellular senescence via BMI1dependent mechanisms, implicating PRC1dependent epigenetics in Ewing’s sarcoma tumorigenesis [13] . Similar to its effects in osteosarcoma, BMI1 drives anchorage-independent Ewing’s sarcoma cell growth in  vitro and tumorigenicity in  vivo through mechanisms that involve BMI1-dependent repression of the adhesion-associated NID-1 and the Hpo tumor suppressive pathway [120,127,128] . These findings strongly implicate PRC1-dependent epigenetics in EWS‑FLI1-induced Ewing’s sarcoma pathogenesis. PRC2 complex in sarcoma

EZH2 is a histone methyltransferase component of the PRC2 Polycomb repression complex that catalyzes H3K27 trimethylation. EZH2 overexpression is linked to several cancers including Hodgkin’s lymphoma and myeloma, and to cancers of the bladder, breast, colon, endometrium, liver, lung, skin, prostate and stomach [129,130] . Transduction of EWS‑FLI1 in primary human MSCs or NESCs induces an Ewing’s sarcoma phenotype and associated Ewing’s sarcoma characteristic gene signature [10,13] . EZH2 is among the genes consistently induced by EWS‑FLI1, and shRNA-mediated EZH2 knockdown suppresses anchorage-independent growth, and dramatically reduces the number and volume of Ewing tumors and lung metastases in mouse xenograft models [7,10] . EWS‑FLI1 effects on PRC2 are solely channeled through EZH2 since ChIP experiments show EZH2 promoter to be 724

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bound by EWS‑FLI1, suggesting direct transcriptional activation mechanisms, and analysis of other PRC2 components show no apparent regulation by EWS‑FLI1 [2,7] . Together, all data indicate that EWS‑FLI1 promotes and maintains an Ewing’s sarcoma program by upregulating the BMI and EZH2 components of the PRC1 and PRC2 complexes. These, in turn, impart Polycomb-regulated epigenomic programs to bypass senescence and block neuroendothelial differentiation in putative cancer stem cells harboring the EWS‑FLI1 fusion [2,7,10,13] . While there are currently no published functional studies on EZH2/PRC2 in other sarcomas, recent reports show that EZH2 is also highly expressed in rhabdomyosarcoma tumors and cell lines, presumably due to low-levels expression of the EZH2-targeting miR-26a miRNA [131] . Moreover, immunohisto­chemical analysis of 32  rhabdomyosarcoma primary tumors showed that EZH2 expression correlates to that of Ki67, a cell proliferation marker and to CD31, an angiogenic marker, establishing a positive correlation between EZH2 expression and rhabdomyosarcoma proliferative and angiogenic indexes [132] . These findings, along with the fact that EZH2 is a potent inhibitor of myogenic differentiation programs in skeletal muscle stem cells [133] , indicate that EZH2/PRC2 epigenetic activities may constitute major oncogenic determinants of rhabdomyosarcoma maintenance and/or genesis. To further test the upregulation of BMI1 and EZH2 in sarcoma, we queried a gene-expression meta-analysis which spans 5372 normal or diseased human tissue samples across 369 different cell and tissue types [134] . Expression of BMI1 (whose product catalyzes H2AK119ub) and of EZH2 (whose product catalyzes H3K27me3) in the 5372 tissues clustered in 15 histological metagroups shows an upregulation of the two HMEs in several cancer metagroups, including sarcoma (p