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EZH2 is induced in hMSC EWS-FLI-1 and expressed in ESFT, supporting tumor cell proliferation. A, EZH2 transcript induction in hMSC EWS-FLI-1 compared with
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Ewing's sarcoma family tumors (ESFT) express the EWS-FLI-1 fusion gene generated by the chromosomal translocation t(11;22)(q24;q12). Expression of the EWS-FLI-1 fusion protein in a permissive cellular environment is believed to play a key role in ESFT pathogenesis. However, EWS-FLI-1 induces growth arrest or apoptosis in differentiated primary cell...
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... for an expected 4.43 ( P = 3.8 E-16). These observations suggest that fibroblasts provide a more restrictive environment than hMSCs for expression of the target gene repertoire of the EWS-FLI-1 fusion protein (17). Importantly, genes that were induced in hMSC EWS-FLI-1 , including STEAP2 , and ID2 , were among the top discriminators for Ewings’ sarcoma (Fig. 3 C ). Among the 12 other sarcomas analyzed, only dermatofibrosarcoma protuberans displayed a gene expression profile with marginal similarity to hMSC ( P = 0.011) (Fig. 3 A and B ). The same statistical analysis was applied to the gene expression profile of hMSC DBDM . In contrast to wt EWS-FLI-1, expression of the EWS-FLI-1 mutant form did not confer on MSCs any statistically significant similarity with Ewing sarcoma (Fig. 3 D ). This observation supports the notion that the lack of shape change displayed by the hMSC DBDM in vitro reflects a different genetic program in these cells, and highlights the importance of DNA binding for the ESFT phenotype–inducing properties of the fusion protein. The highly discriminating ability of the hMSC EWS-FLI-1 gene expression profile for ESFTs was further confirmed by another study, which assessed the transcriptome of a broad range of mesenchymal tumors and identified the CALCB, MAPT , and PRKCB1 genes as prominent Ewing’s sarcoma discriminators (32), all of which we found to be induced in hMSC EWS-FLI-1 (Table 1; Supplementary data 2). Comparison of the genes induced in hMSC EWS-FLI-1 with a set of 38 genes (of which only 34 were included on our microarray) found to be up-regulated in ESFT with respect to a wide spectrum of normal tissues and neuroblastomas (21) revealed 14 shared genes ( P = 1.12e À 15), including the strong ESFT discriminators NPYR1, ITM2A, DKK2, JAK1, STEAP , and EGR2 (Table 1 B ). Moreover, of a subset of 19 transcripts from the 34-gene set that could clearly distinguish an ESFT cell line (SK-N-MC) from neuroblastoma cell lines (21), 8 were part of the hMSC EWS-FLI-1 profile, whereas none of the transcripts in the 34-gene set were identified in the hMSC DBDM expression profile (expected 0.18, P = 1.00). The same study reported that introduction of EWS-FLI-1 into HEK 293 cells induced only one gene of this subset, namely, CCND1 , further highlighting the selective permissiveness of hMSCs for EWS-FLI-1 function (21). Following injection into the subcapsular renal compartment of immunocompromised mice, hMSC EWS-FLI-1 did not form tumors, suggesting that despite expressing many of the hallmarks of ESFT, these cells require some additional event to become tumorigenic in mice. This observation is not surprising in the sense that whereas there have been several examples of a single genetic event transforming mouse progenitor cells (20, 33–35), recent evidence suggests that five events are required to transform human MSCs (36). By analogy to the present study, TLS-ERG and TEL-JAK2 respectively initiated a leukemogenic program and erythropoietin-independent erythropoiesis in normal human hematopoietic cells but fell short of rendering them tumorigenic in vivo (37, 38). EZH2 promotes Ewing’s sarcoma growth. Among transcripts that were up-regulated in hMSC EWS-FLI-1 , we identified the gene encoding EZH2, a member of the polycomb group proteins (Supplementary data 2), which has recently been found to be expressed in ESFT (21). Together with EED and SUZ12, EZH2 forms the polycomb-repressive complex 2, believed to be a key regulator of embryonic development, stem cell renewal, and differentiation (39, 40). EZH2 is the catalytically active component of polycomb- repressive complex 2 and is believed to silence target genes by acting as a methyltransferase specific for Lys 27 of histone H3 and Lys 26 of histone H1 (reviewed in ref. 22). Overexpression of EZH2 has been shown to induce a bypass of the cellular senescence program in mouse embryonic fibroblasts and to prevent mouse hematopoietic stem cell exhaustion (41). Conversely, transient knockdown of EZH2 in primary human fibroblasts and a variety of transformed cells inhibited their proliferation in vitro (42). EZH2 expression has been proposed to be controlled by the Rb-E2F pathway and to be a downstream mediator of E2F-dependent proliferation (42). High EZH2 expression that has been observed in a broad range of tumors has thus far been attributed either to Rb loss or gene amplification (42). Its silencing function is speculated to target tumor suppressor genes (43), but the precise mechanism of its action on cell proliferation has thus far not been elucidated. Because of its role in stem cell maintenance and possibly tumor initiation (42, 44), we addressed the putative implication of EZH2 in ESFT pathogenesis. The EZH2 gene was found to be up- regulated in ESFT compared with normal tissues and neuroblastomas (21), and quantitative real-time PCR analysis confirmed EZH2 induction in hMSC EWS-FLI-1 as well as its dependence on EWS-FLI-1 DBD integrity (Fig. 4 A and data not shown). Expression of EZH2 has been reported to progressively decrease during serial passage of primary fibroblasts and hematopoietic stem cells cultured in vitro , resulting in activation of their senescence program (42). We therefore asked whether the elevated EZH2 expression observed in hMSC EWS-FLI-1 was the result of true up-regulation or mere maintenance of EZH2 transcripts at the level found in low-passage hMSCs. EZH2 expression levels in two different batches of empty vector–, DBDM-, and EWS-FLI-1– infected hMSCs after 20 doublings were compared by real-time PCR with those in parental wt hMSCs after 8 doublings. A 50% decrease in EZH2 expression was observed in cells infected with empty vector and the DBDM that had undergone 20 population doublings compared with parental wt cells that had undergone 8 doublings. By contrast, hMSC EWS-FLI-1 , after 20 doublings, showed a 6- to 7-fold induction of EZH2 expression compared with their 8 population doubling wt counterparts (Fig. 4 A ). This result clearly shows the ability of EWS-FLI-1 to induce EZH2 in hMSCs above the baseline level of their precursors that had undergone fewer population doublings, and is consistent with a possible role in both senescence prevention and stem cell maintenance in hMSCs. We next tested Ewing’s sarcoma samples and xenotransplants of Ewing’s sarcoma cell lines for EZH2 expression and found it to be elevated in both primary Ewing’s sarcoma and ESFT cell line–derived tumors grown in immunocompromised mice (Fig. 4 B ). To address the possible functional implication of EZH2 in Ewing’s sarcoma growth, we relied on a stable shRNA knockdown approach in ESFT cell lines using two distinct RNAi sequences (shRNA1 and shRNA2). Stable EZH2 shRNA expression in A673 and SK-N-MC Ewing’s sarcoma cell lines resulted in >70% reduction of its mRNA level (Fig. 4 C, left ) and a significant decrease in EZH2 protein expression as assessed by Western blot analysis (Fig. 4 C, right ). Reduction of EZH2 expression caused a marked decrease in proliferation of the two cell lines in vitro (Fig. 4 D ). Furthermore, injection of these cells into immunocompromised mice resulted in either no tumor development or strongly reduced tumor growth compared with cells expressing unrelated RNAi sequences (Fig. 5). To exclude possible off-target effects of the shRNA sequences, we repeated these experiments using two additional distinct shRNA sequences and obtained comparable results (data not shown). Based on the notion that EZH2 may repress p16 and possibly other cell cycle regulators (43), we tested the expression level of several cell cycle control genes in the EZH2-depleted cells by real- time PCR. Both of the ESFT cell lines used in our experiments have a nonfunctional p53 pathway (45). The A673 cell line also lacks the p16 INK4A -p14 ARF locus whereas SK-N-MC cells express an inactive Rb protein but retain expression of wt p16 INK4A -p14 ARF (45). Whether or not they were mutated, we did not record any significant change in the expression levels of p14 ARF , p15, p16 INK4A , p18, p21, and p53 following the EZH2 knockdown (data not shown). The observed reduction in proliferation and tumorigenicity can therefore not be explained by an effect on these cell cycle inhibitors and is most likely mediated by a mechanism that has yet to be elucidated. EWS-FLI-1 has thus far been found to induce an oncogenic stress type response in primary human cells, leading to cell cycle arrest, and a partial ESFT transcriptome in a variety of heterologous cell lines. Our present observations identify MSCs as the first primary human cell type that, while accurately recapitulating the ESFT gene expression profile, maintains viability and proliferation in response to EWS-FLI-1. EWS-FLI-1 induced marked rounding of hMSCs in vitro that accompanied robust expression changes of genes implicated in cell differentiation. Consistent with previous studies in tumor cells and immortalized fibroblasts, microarray analysis revealed that numerous genes induced in hMSC EWS-FLI-1 are implicated in neural crest development and neuronal differentiation. Among the most differentially expressed transcripts, we found NGFR (p75), which was strongly induced at both the transcription and protein levels, and which, in addition to playing a central role in neural develop- ment, is a key marker of neuroectodermal stem cells in both normal tissues and neural crest–derived tumors (46). Among the other genes that constitute part of the neuroectodermal profile of hMSC EWS-FLI-1 , SOX2 has been shown to maintain neural progenitor features (47). In contrast to observations in mouse mesenchymal progenitor cells, which were reported to lose osteogenic and adipocytic differentiation potential as a result of EWS-FLI-1 expression, hMSCs expressing EWS-FLI-1 retained at least some degree of trilineage differentiation plasticity. It is plausible that the early- stage neuroectodermal ...
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... which only 34 were included on our microarray) found to be up-regulated in ESFT with respect to a wide spectrum of normal tissues and neuroblastomas (21) revealed 14 shared genes ( P = 1.12e À 15), including the strong ESFT discriminators NPYR1, ITM2A, DKK2, JAK1, STEAP , and EGR2 (Table 1 B ). Moreover, of a subset of 19 transcripts from the 34-gene set that could clearly distinguish an ESFT cell line (SK-N-MC) from neuroblastoma cell lines (21), 8 were part of the hMSC EWS-FLI-1 profile, whereas none of the transcripts in the 34-gene set were identified in the hMSC DBDM expression profile (expected 0.18, P = 1.00). The same study reported that introduction of EWS-FLI-1 into HEK 293 cells induced only one gene of this subset, namely, CCND1 , further highlighting the selective permissiveness of hMSCs for EWS-FLI-1 function (21). Following injection into the subcapsular renal compartment of immunocompromised mice, hMSC EWS-FLI-1 did not form tumors, suggesting that despite expressing many of the hallmarks of ESFT, these cells require some additional event to become tumorigenic in mice. This observation is not surprising in the sense that whereas there have been several examples of a single genetic event transforming mouse progenitor cells (20, 33–35), recent evidence suggests that five events are required to transform human MSCs (36). By analogy to the present study, TLS-ERG and TEL-JAK2 respectively initiated a leukemogenic program and erythropoietin-independent erythropoiesis in normal human hematopoietic cells but fell short of rendering them tumorigenic in vivo (37, 38). EZH2 promotes Ewing’s sarcoma growth. Among transcripts that were up-regulated in hMSC EWS-FLI-1 , we identified the gene encoding EZH2, a member of the polycomb group proteins (Supplementary data 2), which has recently been found to be expressed in ESFT (21). Together with EED and SUZ12, EZH2 forms the polycomb-repressive complex 2, believed to be a key regulator of embryonic development, stem cell renewal, and differentiation (39, 40). EZH2 is the catalytically active component of polycomb- repressive complex 2 and is believed to silence target genes by acting as a methyltransferase specific for Lys 27 of histone H3 and Lys 26 of histone H1 (reviewed in ref. 22). Overexpression of EZH2 has been shown to induce a bypass of the cellular senescence program in mouse embryonic fibroblasts and to prevent mouse hematopoietic stem cell exhaustion (41). Conversely, transient knockdown of EZH2 in primary human fibroblasts and a variety of transformed cells inhibited their proliferation in vitro (42). EZH2 expression has been proposed to be controlled by the Rb-E2F pathway and to be a downstream mediator of E2F-dependent proliferation (42). High EZH2 expression that has been observed in a broad range of tumors has thus far been attributed either to Rb loss or gene amplification (42). Its silencing function is speculated to target tumor suppressor genes (43), but the precise mechanism of its action on cell proliferation has thus far not been elucidated. Because of its role in stem cell maintenance and possibly tumor initiation (42, 44), we addressed the putative implication of EZH2 in ESFT pathogenesis. The EZH2 gene was found to be up- regulated in ESFT compared with normal tissues and neuroblastomas (21), and quantitative real-time PCR analysis confirmed EZH2 induction in hMSC EWS-FLI-1 as well as its dependence on EWS-FLI-1 DBD integrity (Fig. 4 A and data not shown). Expression of EZH2 has been reported to progressively decrease during serial passage of primary fibroblasts and hematopoietic stem cells cultured in vitro , resulting in activation of their senescence program (42). We therefore asked whether the elevated EZH2 expression observed in hMSC EWS-FLI-1 was the result of true up-regulation or mere maintenance of EZH2 transcripts at the level found in low-passage hMSCs. EZH2 expression levels in two different batches of empty vector–, DBDM-, and EWS-FLI-1– infected hMSCs after 20 doublings were compared by real-time PCR with those in parental wt hMSCs after 8 doublings. A 50% decrease in EZH2 expression was observed in cells infected with empty vector and the DBDM that had undergone 20 population doublings compared with parental wt cells that had undergone 8 doublings. By contrast, hMSC EWS-FLI-1 , after 20 doublings, showed a 6- to 7-fold induction of EZH2 expression compared with their 8 population doubling wt counterparts (Fig. 4 A ). This result clearly shows the ability of EWS-FLI-1 to induce EZH2 in hMSCs above the baseline level of their precursors that had undergone fewer population doublings, and is consistent with a possible role in both senescence prevention and stem cell maintenance in hMSCs. We next tested Ewing’s sarcoma samples and xenotransplants of Ewing’s sarcoma cell lines for EZH2 expression and found it to be elevated in both primary Ewing’s sarcoma and ESFT cell line–derived tumors grown in immunocompromised mice (Fig. 4 B ). To address the possible functional implication of EZH2 in Ewing’s sarcoma growth, we relied on a stable shRNA knockdown approach in ESFT cell lines using two distinct RNAi sequences (shRNA1 and shRNA2). Stable EZH2 shRNA expression in A673 and SK-N-MC Ewing’s sarcoma cell lines resulted in >70% reduction of its mRNA level (Fig. 4 C, left ) and a significant decrease in EZH2 protein expression as assessed by Western blot analysis (Fig. 4 C, right ). Reduction of EZH2 expression caused a marked decrease in proliferation of the two cell lines in vitro (Fig. 4 D ). Furthermore, injection of these cells into immunocompromised mice resulted in either no tumor development or strongly reduced tumor growth compared with cells expressing unrelated RNAi sequences (Fig. 5). To exclude possible off-target effects of the shRNA sequences, we repeated these experiments using two additional distinct shRNA sequences and obtained comparable results (data not shown). Based on the notion that EZH2 may repress p16 and possibly other cell cycle regulators (43), we tested the expression level of several cell cycle control genes in the EZH2-depleted cells by real- time PCR. Both of the ESFT cell lines used in our experiments have a nonfunctional p53 pathway (45). The A673 cell line also lacks the p16 INK4A -p14 ARF locus whereas SK-N-MC cells express an inactive Rb protein but retain expression of wt p16 INK4A -p14 ARF (45). Whether or not they were mutated, we did not record any significant change in the expression levels of p14 ARF , p15, p16 INK4A , p18, p21, and p53 following the EZH2 knockdown (data not shown). The observed reduction in proliferation and tumorigenicity can therefore not be explained by an effect on these cell cycle inhibitors and is most likely mediated by a mechanism that has yet to be elucidated. EWS-FLI-1 has thus far been found to induce an oncogenic stress type response in primary human cells, leading to cell cycle arrest, and a partial ESFT transcriptome in a variety of heterologous cell lines. Our present observations identify MSCs as the first primary human cell type that, while accurately recapitulating the ESFT gene expression profile, maintains viability and proliferation in response to EWS-FLI-1. EWS-FLI-1 induced marked rounding of hMSCs in vitro that accompanied robust expression changes of genes implicated in cell differentiation. Consistent with previous studies in tumor cells and immortalized fibroblasts, microarray analysis revealed that numerous genes induced in hMSC EWS-FLI-1 are implicated in neural crest development and neuronal differentiation. Among the most differentially expressed transcripts, we found NGFR (p75), which was strongly induced at both the transcription and protein levels, and which, in addition to playing a central role in neural develop- ment, is a key marker of neuroectodermal stem cells in both normal tissues and neural crest–derived tumors (46). Among the other genes that constitute part of the neuroectodermal profile of hMSC EWS-FLI-1 , SOX2 has been shown to maintain neural progenitor features (47). In contrast to observations in mouse mesenchymal progenitor cells, which were reported to lose osteogenic and adipocytic differentiation potential as a result of EWS-FLI-1 expression, hMSCs expressing EWS-FLI-1 retained at least some degree of trilineage differentiation plasticity. It is plausible that the early- stage neuroectodermal differentiation program induced by EWS- FLI-1 in hMSCs can still be overridden by the supraphysiologic conditions of the in vitro differentiation assays. Some of the genes that are implicated in neural differentiation have recently been suggested to play an active role in ESFT pathogenesis. Thus, NROB1 can promote tumorigenesis of Ewing’s sarcoma cell lines (7), whereas NKX2-2 may provide important functions at specific stages of ESFT development because its repression strongly reduced ESFT cell line tumorigenicity (10). It is noteworthy that both genes were not only strongly induced in hMSC EWS-FLI-1 but also remained dependent on EWS-FLI-1 expression in established and tumorigenic ESFT cell lines. These observations suggest that they play a role not only during the initial steps of tumor development but also at late stages of tumor growth. Importantly, several of the above genes have been found to be strong discriminators of ESFT, and their expression has been used as an argument that ESFT may be of neuroectodermal origin. However, the present experiments show that expression of genes implicated in neuronal differentiation and neural crest development can be induced by EWS-FLI-1 in the appropriate primary mesenchymal progenitor cell environment. Thus, a hMSC that can undergo partial neuroectodermal differentiation may constitute the origin of ESFT, suggesting that these tumors need not arise from a neuroectodermal precursor to explain their primitive neuroectodermal phenotype. Several of the genes observed to be ...
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... from the 34-gene set that could clearly distinguish an ESFT cell line (SK-N-MC) from neuroblastoma cell lines (21), 8 were part of the hMSC EWS-FLI-1 profile, whereas none of the transcripts in the 34-gene set were identified in the hMSC DBDM expression profile (expected 0.18, P = 1.00). The same study reported that introduction of EWS-FLI-1 into HEK 293 cells induced only one gene of this subset, namely, CCND1 , further highlighting the selective permissiveness of hMSCs for EWS-FLI-1 function (21). Following injection into the subcapsular renal compartment of immunocompromised mice, hMSC EWS-FLI-1 did not form tumors, suggesting that despite expressing many of the hallmarks of ESFT, these cells require some additional event to become tumorigenic in mice. This observation is not surprising in the sense that whereas there have been several examples of a single genetic event transforming mouse progenitor cells (20, 33–35), recent evidence suggests that five events are required to transform human MSCs (36). By analogy to the present study, TLS-ERG and TEL-JAK2 respectively initiated a leukemogenic program and erythropoietin-independent erythropoiesis in normal human hematopoietic cells but fell short of rendering them tumorigenic in vivo (37, 38). EZH2 promotes Ewing’s sarcoma growth. Among transcripts that were up-regulated in hMSC EWS-FLI-1 , we identified the gene encoding EZH2, a member of the polycomb group proteins (Supplementary data 2), which has recently been found to be expressed in ESFT (21). Together with EED and SUZ12, EZH2 forms the polycomb-repressive complex 2, believed to be a key regulator of embryonic development, stem cell renewal, and differentiation (39, 40). EZH2 is the catalytically active component of polycomb- repressive complex 2 and is believed to silence target genes by acting as a methyltransferase specific for Lys 27 of histone H3 and Lys 26 of histone H1 (reviewed in ref. 22). Overexpression of EZH2 has been shown to induce a bypass of the cellular senescence program in mouse embryonic fibroblasts and to prevent mouse hematopoietic stem cell exhaustion (41). Conversely, transient knockdown of EZH2 in primary human fibroblasts and a variety of transformed cells inhibited their proliferation in vitro (42). EZH2 expression has been proposed to be controlled by the Rb-E2F pathway and to be a downstream mediator of E2F-dependent proliferation (42). High EZH2 expression that has been observed in a broad range of tumors has thus far been attributed either to Rb loss or gene amplification (42). Its silencing function is speculated to target tumor suppressor genes (43), but the precise mechanism of its action on cell proliferation has thus far not been elucidated. Because of its role in stem cell maintenance and possibly tumor initiation (42, 44), we addressed the putative implication of EZH2 in ESFT pathogenesis. The EZH2 gene was found to be up- regulated in ESFT compared with normal tissues and neuroblastomas (21), and quantitative real-time PCR analysis confirmed EZH2 induction in hMSC EWS-FLI-1 as well as its dependence on EWS-FLI-1 DBD integrity (Fig. 4 A and data not shown). Expression of EZH2 has been reported to progressively decrease during serial passage of primary fibroblasts and hematopoietic stem cells cultured in vitro , resulting in activation of their senescence program (42). We therefore asked whether the elevated EZH2 expression observed in hMSC EWS-FLI-1 was the result of true up-regulation or mere maintenance of EZH2 transcripts at the level found in low-passage hMSCs. EZH2 expression levels in two different batches of empty vector–, DBDM-, and EWS-FLI-1– infected hMSCs after 20 doublings were compared by real-time PCR with those in parental wt hMSCs after 8 doublings. A 50% decrease in EZH2 expression was observed in cells infected with empty vector and the DBDM that had undergone 20 population doublings compared with parental wt cells that had undergone 8 doublings. By contrast, hMSC EWS-FLI-1 , after 20 doublings, showed a 6- to 7-fold induction of EZH2 expression compared with their 8 population doubling wt counterparts (Fig. 4 A ). This result clearly shows the ability of EWS-FLI-1 to induce EZH2 in hMSCs above the baseline level of their precursors that had undergone fewer population doublings, and is consistent with a possible role in both senescence prevention and stem cell maintenance in hMSCs. We next tested Ewing’s sarcoma samples and xenotransplants of Ewing’s sarcoma cell lines for EZH2 expression and found it to be elevated in both primary Ewing’s sarcoma and ESFT cell line–derived tumors grown in immunocompromised mice (Fig. 4 B ). To address the possible functional implication of EZH2 in Ewing’s sarcoma growth, we relied on a stable shRNA knockdown approach in ESFT cell lines using two distinct RNAi sequences (shRNA1 and shRNA2). Stable EZH2 shRNA expression in A673 and SK-N-MC Ewing’s sarcoma cell lines resulted in >70% reduction of its mRNA level (Fig. 4 C, left ) and a significant decrease in EZH2 protein expression as assessed by Western blot analysis (Fig. 4 C, right ). Reduction of EZH2 expression caused a marked decrease in proliferation of the two cell lines in vitro (Fig. 4 D ). Furthermore, injection of these cells into immunocompromised mice resulted in either no tumor development or strongly reduced tumor growth compared with cells expressing unrelated RNAi sequences (Fig. 5). To exclude possible off-target effects of the shRNA sequences, we repeated these experiments using two additional distinct shRNA sequences and obtained comparable results (data not shown). Based on the notion that EZH2 may repress p16 and possibly other cell cycle regulators (43), we tested the expression level of several cell cycle control genes in the EZH2-depleted cells by real- time PCR. Both of the ESFT cell lines used in our experiments have a nonfunctional p53 pathway (45). The A673 cell line also lacks the p16 INK4A -p14 ARF locus whereas SK-N-MC cells express an inactive Rb protein but retain expression of wt p16 INK4A -p14 ARF (45). Whether or not they were mutated, we did not record any significant change in the expression levels of p14 ARF , p15, p16 INK4A , p18, p21, and p53 following the EZH2 knockdown (data not shown). The observed reduction in proliferation and tumorigenicity can therefore not be explained by an effect on these cell cycle inhibitors and is most likely mediated by a mechanism that has yet to be elucidated. EWS-FLI-1 has thus far been found to induce an oncogenic stress type response in primary human cells, leading to cell cycle arrest, and a partial ESFT transcriptome in a variety of heterologous cell lines. Our present observations identify MSCs as the first primary human cell type that, while accurately recapitulating the ESFT gene expression profile, maintains viability and proliferation in response to EWS-FLI-1. EWS-FLI-1 induced marked rounding of hMSCs in vitro that accompanied robust expression changes of genes implicated in cell differentiation. Consistent with previous studies in tumor cells and immortalized fibroblasts, microarray analysis revealed that numerous genes induced in hMSC EWS-FLI-1 are implicated in neural crest development and neuronal differentiation. Among the most differentially expressed transcripts, we found NGFR (p75), which was strongly induced at both the transcription and protein levels, and which, in addition to playing a central role in neural develop- ment, is a key marker of neuroectodermal stem cells in both normal tissues and neural crest–derived tumors (46). Among the other genes that constitute part of the neuroectodermal profile of hMSC EWS-FLI-1 , SOX2 has been shown to maintain neural progenitor features (47). In contrast to observations in mouse mesenchymal progenitor cells, which were reported to lose osteogenic and adipocytic differentiation potential as a result of EWS-FLI-1 expression, hMSCs expressing EWS-FLI-1 retained at least some degree of trilineage differentiation plasticity. It is plausible that the early- stage neuroectodermal differentiation program induced by EWS- FLI-1 in hMSCs can still be overridden by the supraphysiologic conditions of the in vitro differentiation assays. Some of the genes that are implicated in neural differentiation have recently been suggested to play an active role in ESFT pathogenesis. Thus, NROB1 can promote tumorigenesis of Ewing’s sarcoma cell lines (7), whereas NKX2-2 may provide important functions at specific stages of ESFT development because its repression strongly reduced ESFT cell line tumorigenicity (10). It is noteworthy that both genes were not only strongly induced in hMSC EWS-FLI-1 but also remained dependent on EWS-FLI-1 expression in established and tumorigenic ESFT cell lines. These observations suggest that they play a role not only during the initial steps of tumor development but also at late stages of tumor growth. Importantly, several of the above genes have been found to be strong discriminators of ESFT, and their expression has been used as an argument that ESFT may be of neuroectodermal origin. However, the present experiments show that expression of genes implicated in neuronal differentiation and neural crest development can be induced by EWS-FLI-1 in the appropriate primary mesenchymal progenitor cell environment. Thus, a hMSC that can undergo partial neuroectodermal differentiation may constitute the origin of ESFT, suggesting that these tumors need not arise from a neuroectodermal precursor to explain their primitive neuroectodermal phenotype. Several of the genes observed to be up-regulated in hMSC EWS-FLI-1 and expressed in ESFT have been proposed to be functionally related to Ewings’ sarcoma development and behavior. Thus, ID2 , observed to be induced by EWS-FLI-1 in both hMSCs and mouse mesenchymal progenitor cells (20), may play an important role in promoting cell cycle entry by inhibiting Rb. Induction of fos ...
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... phenotype–inducing properties of the fusion protein. The highly discriminating ability of the hMSC EWS-FLI-1 gene expression profile for ESFTs was further confirmed by another study, which assessed the transcriptome of a broad range of mesenchymal tumors and identified the CALCB, MAPT , and PRKCB1 genes as prominent Ewing’s sarcoma discriminators (32), all of which we found to be induced in hMSC EWS-FLI-1 (Table 1; Supplementary data 2). Comparison of the genes induced in hMSC EWS-FLI-1 with a set of 38 genes (of which only 34 were included on our microarray) found to be up-regulated in ESFT with respect to a wide spectrum of normal tissues and neuroblastomas (21) revealed 14 shared genes ( P = 1.12e À 15), including the strong ESFT discriminators NPYR1, ITM2A, DKK2, JAK1, STEAP , and EGR2 (Table 1 B ). Moreover, of a subset of 19 transcripts from the 34-gene set that could clearly distinguish an ESFT cell line (SK-N-MC) from neuroblastoma cell lines (21), 8 were part of the hMSC EWS-FLI-1 profile, whereas none of the transcripts in the 34-gene set were identified in the hMSC DBDM expression profile (expected 0.18, P = 1.00). The same study reported that introduction of EWS-FLI-1 into HEK 293 cells induced only one gene of this subset, namely, CCND1 , further highlighting the selective permissiveness of hMSCs for EWS-FLI-1 function (21). Following injection into the subcapsular renal compartment of immunocompromised mice, hMSC EWS-FLI-1 did not form tumors, suggesting that despite expressing many of the hallmarks of ESFT, these cells require some additional event to become tumorigenic in mice. This observation is not surprising in the sense that whereas there have been several examples of a single genetic event transforming mouse progenitor cells (20, 33–35), recent evidence suggests that five events are required to transform human MSCs (36). By analogy to the present study, TLS-ERG and TEL-JAK2 respectively initiated a leukemogenic program and erythropoietin-independent erythropoiesis in normal human hematopoietic cells but fell short of rendering them tumorigenic in vivo (37, 38). EZH2 promotes Ewing’s sarcoma growth. Among transcripts that were up-regulated in hMSC EWS-FLI-1 , we identified the gene encoding EZH2, a member of the polycomb group proteins (Supplementary data 2), which has recently been found to be expressed in ESFT (21). Together with EED and SUZ12, EZH2 forms the polycomb-repressive complex 2, believed to be a key regulator of embryonic development, stem cell renewal, and differentiation (39, 40). EZH2 is the catalytically active component of polycomb- repressive complex 2 and is believed to silence target genes by acting as a methyltransferase specific for Lys 27 of histone H3 and Lys 26 of histone H1 (reviewed in ref. 22). Overexpression of EZH2 has been shown to induce a bypass of the cellular senescence program in mouse embryonic fibroblasts and to prevent mouse hematopoietic stem cell exhaustion (41). Conversely, transient knockdown of EZH2 in primary human fibroblasts and a variety of transformed cells inhibited their proliferation in vitro (42). EZH2 expression has been proposed to be controlled by the Rb-E2F pathway and to be a downstream mediator of E2F-dependent proliferation (42). High EZH2 expression that has been observed in a broad range of tumors has thus far been attributed either to Rb loss or gene amplification (42). Its silencing function is speculated to target tumor suppressor genes (43), but the precise mechanism of its action on cell proliferation has thus far not been elucidated. Because of its role in stem cell maintenance and possibly tumor initiation (42, 44), we addressed the putative implication of EZH2 in ESFT pathogenesis. The EZH2 gene was found to be up- regulated in ESFT compared with normal tissues and neuroblastomas (21), and quantitative real-time PCR analysis confirmed EZH2 induction in hMSC EWS-FLI-1 as well as its dependence on EWS-FLI-1 DBD integrity (Fig. 4 A and data not shown). Expression of EZH2 has been reported to progressively decrease during serial passage of primary fibroblasts and hematopoietic stem cells cultured in vitro , resulting in activation of their senescence program (42). We therefore asked whether the elevated EZH2 expression observed in hMSC EWS-FLI-1 was the result of true up-regulation or mere maintenance of EZH2 transcripts at the level found in low-passage hMSCs. EZH2 expression levels in two different batches of empty vector–, DBDM-, and EWS-FLI-1– infected hMSCs after 20 doublings were compared by real-time PCR with those in parental wt hMSCs after 8 doublings. A 50% decrease in EZH2 expression was observed in cells infected with empty vector and the DBDM that had undergone 20 population doublings compared with parental wt cells that had undergone 8 doublings. By contrast, hMSC EWS-FLI-1 , after 20 doublings, showed a 6- to 7-fold induction of EZH2 expression compared with their 8 population doubling wt counterparts (Fig. 4 A ). This result clearly shows the ability of EWS-FLI-1 to induce EZH2 in hMSCs above the baseline level of their precursors that had undergone fewer population doublings, and is consistent with a possible role in both senescence prevention and stem cell maintenance in hMSCs. We next tested Ewing’s sarcoma samples and xenotransplants of Ewing’s sarcoma cell lines for EZH2 expression and found it to be elevated in both primary Ewing’s sarcoma and ESFT cell line–derived tumors grown in immunocompromised mice (Fig. 4 B ). To address the possible functional implication of EZH2 in Ewing’s sarcoma growth, we relied on a stable shRNA knockdown approach in ESFT cell lines using two distinct RNAi sequences (shRNA1 and shRNA2). Stable EZH2 shRNA expression in A673 and SK-N-MC Ewing’s sarcoma cell lines resulted in >70% reduction of its mRNA level (Fig. 4 C, left ) and a significant decrease in EZH2 protein expression as assessed by Western blot analysis (Fig. 4 C, right ). Reduction of EZH2 expression caused a marked decrease in proliferation of the two cell lines in vitro (Fig. 4 D ). Furthermore, injection of these cells into immunocompromised mice resulted in either no tumor development or strongly reduced tumor growth compared with cells expressing unrelated RNAi sequences (Fig. 5). To exclude possible off-target effects of the shRNA sequences, we repeated these experiments using two additional distinct shRNA sequences and obtained comparable results (data not shown). Based on the notion that EZH2 may repress p16 and possibly other cell cycle regulators (43), we tested the expression level of several cell cycle control genes in the EZH2-depleted cells by real- time PCR. Both of the ESFT cell lines used in our experiments have a nonfunctional p53 pathway (45). The A673 cell line also lacks the p16 INK4A -p14 ARF locus whereas SK-N-MC cells express an inactive Rb protein but retain expression of wt p16 INK4A -p14 ARF (45). Whether or not they were mutated, we did not record any significant change in the expression levels of p14 ARF , p15, p16 INK4A , p18, p21, and p53 following the EZH2 knockdown (data not shown). The observed reduction in proliferation and tumorigenicity can therefore not be explained by an effect on these cell cycle inhibitors and is most likely mediated by a mechanism that has yet to be elucidated. EWS-FLI-1 has thus far been found to induce an oncogenic stress type response in primary human cells, leading to cell cycle arrest, and a partial ESFT transcriptome in a variety of heterologous cell lines. Our present observations identify MSCs as the first primary human cell type that, while accurately recapitulating the ESFT gene expression profile, maintains viability and proliferation in response to EWS-FLI-1. EWS-FLI-1 induced marked rounding of hMSCs in vitro that accompanied robust expression changes of genes implicated in cell differentiation. Consistent with previous studies in tumor cells and immortalized fibroblasts, microarray analysis revealed that numerous genes induced in hMSC EWS-FLI-1 are implicated in neural crest development and neuronal differentiation. Among the most differentially expressed transcripts, we found NGFR (p75), which was strongly induced at both the transcription and protein levels, and which, in addition to playing a central role in neural develop- ment, is a key marker of neuroectodermal stem cells in both normal tissues and neural crest–derived tumors (46). Among the other genes that constitute part of the neuroectodermal profile of hMSC EWS-FLI-1 , SOX2 has been shown to maintain neural progenitor features (47). In contrast to observations in mouse mesenchymal progenitor cells, which were reported to lose osteogenic and adipocytic differentiation potential as a result of EWS-FLI-1 expression, hMSCs expressing EWS-FLI-1 retained at least some degree of trilineage differentiation plasticity. It is plausible that the early- stage neuroectodermal differentiation program induced by EWS- FLI-1 in hMSCs can still be overridden by the supraphysiologic conditions of the in vitro differentiation assays. Some of the genes that are implicated in neural differentiation have recently been suggested to play an active role in ESFT pathogenesis. Thus, NROB1 can promote tumorigenesis of Ewing’s sarcoma cell lines (7), whereas NKX2-2 may provide important functions at specific stages of ESFT development because its repression strongly reduced ESFT cell line tumorigenicity (10). It is noteworthy that both genes were not only strongly induced in hMSC EWS-FLI-1 but also remained dependent on EWS-FLI-1 expression in established and tumorigenic ESFT cell lines. These observations suggest that they play a role not only during the initial steps of tumor development but also at late stages of tumor growth. Importantly, several of the above genes have been found to be strong discriminators of ESFT, and their expression has been used as an argument that ESFT may be of ...
Context 5
... (21), 8 were part of the hMSC EWS-FLI-1 profile, whereas none of the transcripts in the 34-gene set were identified in the hMSC DBDM expression profile (expected 0.18, P = 1.00). The same study reported that introduction of EWS-FLI-1 into HEK 293 cells induced only one gene of this subset, namely, CCND1 , further highlighting the selective permissiveness of hMSCs for EWS-FLI-1 function (21). Following injection into the subcapsular renal compartment of immunocompromised mice, hMSC EWS-FLI-1 did not form tumors, suggesting that despite expressing many of the hallmarks of ESFT, these cells require some additional event to become tumorigenic in mice. This observation is not surprising in the sense that whereas there have been several examples of a single genetic event transforming mouse progenitor cells (20, 33–35), recent evidence suggests that five events are required to transform human MSCs (36). By analogy to the present study, TLS-ERG and TEL-JAK2 respectively initiated a leukemogenic program and erythropoietin-independent erythropoiesis in normal human hematopoietic cells but fell short of rendering them tumorigenic in vivo (37, 38). EZH2 promotes Ewing’s sarcoma growth. Among transcripts that were up-regulated in hMSC EWS-FLI-1 , we identified the gene encoding EZH2, a member of the polycomb group proteins (Supplementary data 2), which has recently been found to be expressed in ESFT (21). Together with EED and SUZ12, EZH2 forms the polycomb-repressive complex 2, believed to be a key regulator of embryonic development, stem cell renewal, and differentiation (39, 40). EZH2 is the catalytically active component of polycomb- repressive complex 2 and is believed to silence target genes by acting as a methyltransferase specific for Lys 27 of histone H3 and Lys 26 of histone H1 (reviewed in ref. 22). Overexpression of EZH2 has been shown to induce a bypass of the cellular senescence program in mouse embryonic fibroblasts and to prevent mouse hematopoietic stem cell exhaustion (41). Conversely, transient knockdown of EZH2 in primary human fibroblasts and a variety of transformed cells inhibited their proliferation in vitro (42). EZH2 expression has been proposed to be controlled by the Rb-E2F pathway and to be a downstream mediator of E2F-dependent proliferation (42). High EZH2 expression that has been observed in a broad range of tumors has thus far been attributed either to Rb loss or gene amplification (42). Its silencing function is speculated to target tumor suppressor genes (43), but the precise mechanism of its action on cell proliferation has thus far not been elucidated. Because of its role in stem cell maintenance and possibly tumor initiation (42, 44), we addressed the putative implication of EZH2 in ESFT pathogenesis. The EZH2 gene was found to be up- regulated in ESFT compared with normal tissues and neuroblastomas (21), and quantitative real-time PCR analysis confirmed EZH2 induction in hMSC EWS-FLI-1 as well as its dependence on EWS-FLI-1 DBD integrity (Fig. 4 A and data not shown). Expression of EZH2 has been reported to progressively decrease during serial passage of primary fibroblasts and hematopoietic stem cells cultured in vitro , resulting in activation of their senescence program (42). We therefore asked whether the elevated EZH2 expression observed in hMSC EWS-FLI-1 was the result of true up-regulation or mere maintenance of EZH2 transcripts at the level found in low-passage hMSCs. EZH2 expression levels in two different batches of empty vector–, DBDM-, and EWS-FLI-1– infected hMSCs after 20 doublings were compared by real-time PCR with those in parental wt hMSCs after 8 doublings. A 50% decrease in EZH2 expression was observed in cells infected with empty vector and the DBDM that had undergone 20 population doublings compared with parental wt cells that had undergone 8 doublings. By contrast, hMSC EWS-FLI-1 , after 20 doublings, showed a 6- to 7-fold induction of EZH2 expression compared with their 8 population doubling wt counterparts (Fig. 4 A ). This result clearly shows the ability of EWS-FLI-1 to induce EZH2 in hMSCs above the baseline level of their precursors that had undergone fewer population doublings, and is consistent with a possible role in both senescence prevention and stem cell maintenance in hMSCs. We next tested Ewing’s sarcoma samples and xenotransplants of Ewing’s sarcoma cell lines for EZH2 expression and found it to be elevated in both primary Ewing’s sarcoma and ESFT cell line–derived tumors grown in immunocompromised mice (Fig. 4 B ). To address the possible functional implication of EZH2 in Ewing’s sarcoma growth, we relied on a stable shRNA knockdown approach in ESFT cell lines using two distinct RNAi sequences (shRNA1 and shRNA2). Stable EZH2 shRNA expression in A673 and SK-N-MC Ewing’s sarcoma cell lines resulted in >70% reduction of its mRNA level (Fig. 4 C, left ) and a significant decrease in EZH2 protein expression as assessed by Western blot analysis (Fig. 4 C, right ). Reduction of EZH2 expression caused a marked decrease in proliferation of the two cell lines in vitro (Fig. 4 D ). Furthermore, injection of these cells into immunocompromised mice resulted in either no tumor development or strongly reduced tumor growth compared with cells expressing unrelated RNAi sequences (Fig. 5). To exclude possible off-target effects of the shRNA sequences, we repeated these experiments using two additional distinct shRNA sequences and obtained comparable results (data not shown). Based on the notion that EZH2 may repress p16 and possibly other cell cycle regulators (43), we tested the expression level of several cell cycle control genes in the EZH2-depleted cells by real- time PCR. Both of the ESFT cell lines used in our experiments have a nonfunctional p53 pathway (45). The A673 cell line also lacks the p16 INK4A -p14 ARF locus whereas SK-N-MC cells express an inactive Rb protein but retain expression of wt p16 INK4A -p14 ARF (45). Whether or not they were mutated, we did not record any significant change in the expression levels of p14 ARF , p15, p16 INK4A , p18, p21, and p53 following the EZH2 knockdown (data not shown). The observed reduction in proliferation and tumorigenicity can therefore not be explained by an effect on these cell cycle inhibitors and is most likely mediated by a mechanism that has yet to be elucidated. EWS-FLI-1 has thus far been found to induce an oncogenic stress type response in primary human cells, leading to cell cycle arrest, and a partial ESFT transcriptome in a variety of heterologous cell lines. Our present observations identify MSCs as the first primary human cell type that, while accurately recapitulating the ESFT gene expression profile, maintains viability and proliferation in response to EWS-FLI-1. EWS-FLI-1 induced marked rounding of hMSCs in vitro that accompanied robust expression changes of genes implicated in cell differentiation. Consistent with previous studies in tumor cells and immortalized fibroblasts, microarray analysis revealed that numerous genes induced in hMSC EWS-FLI-1 are implicated in neural crest development and neuronal differentiation. Among the most differentially expressed transcripts, we found NGFR (p75), which was strongly induced at both the transcription and protein levels, and which, in addition to playing a central role in neural develop- ment, is a key marker of neuroectodermal stem cells in both normal tissues and neural crest–derived tumors (46). Among the other genes that constitute part of the neuroectodermal profile of hMSC EWS-FLI-1 , SOX2 has been shown to maintain neural progenitor features (47). In contrast to observations in mouse mesenchymal progenitor cells, which were reported to lose osteogenic and adipocytic differentiation potential as a result of EWS-FLI-1 expression, hMSCs expressing EWS-FLI-1 retained at least some degree of trilineage differentiation plasticity. It is plausible that the early- stage neuroectodermal differentiation program induced by EWS- FLI-1 in hMSCs can still be overridden by the supraphysiologic conditions of the in vitro differentiation assays. Some of the genes that are implicated in neural differentiation have recently been suggested to play an active role in ESFT pathogenesis. Thus, NROB1 can promote tumorigenesis of Ewing’s sarcoma cell lines (7), whereas NKX2-2 may provide important functions at specific stages of ESFT development because its repression strongly reduced ESFT cell line tumorigenicity (10). It is noteworthy that both genes were not only strongly induced in hMSC EWS-FLI-1 but also remained dependent on EWS-FLI-1 expression in established and tumorigenic ESFT cell lines. These observations suggest that they play a role not only during the initial steps of tumor development but also at late stages of tumor growth. Importantly, several of the above genes have been found to be strong discriminators of ESFT, and their expression has been used as an argument that ESFT may be of neuroectodermal origin. However, the present experiments show that expression of genes implicated in neuronal differentiation and neural crest development can be induced by EWS-FLI-1 in the appropriate primary mesenchymal progenitor cell environment. Thus, a hMSC that can undergo partial neuroectodermal differentiation may constitute the origin of ESFT, suggesting that these tumors need not arise from a neuroectodermal precursor to explain their primitive neuroectodermal phenotype. Several of the genes observed to be up-regulated in hMSC EWS-FLI-1 and expressed in ESFT have been proposed to be functionally related to Ewings’ sarcoma development and behavior. Thus, ID2 , observed to be induced by EWS-FLI-1 in both hMSCs and mouse mesenchymal progenitor cells (20), may play an important role in promoting cell cycle entry by inhibiting Rb. Induction of fos that was detected as a result of EWS-FLI-1 introduction into hMSCs (Supplementary data 1C) may be of ...
Context 6
... hMSC DBDM expression profile (expected 0.18, P = 1.00). The same study reported that introduction of EWS-FLI-1 into HEK 293 cells induced only one gene of this subset, namely, CCND1 , further highlighting the selective permissiveness of hMSCs for EWS-FLI-1 function (21). Following injection into the subcapsular renal compartment of immunocompromised mice, hMSC EWS-FLI-1 did not form tumors, suggesting that despite expressing many of the hallmarks of ESFT, these cells require some additional event to become tumorigenic in mice. This observation is not surprising in the sense that whereas there have been several examples of a single genetic event transforming mouse progenitor cells (20, 33–35), recent evidence suggests that five events are required to transform human MSCs (36). By analogy to the present study, TLS-ERG and TEL-JAK2 respectively initiated a leukemogenic program and erythropoietin-independent erythropoiesis in normal human hematopoietic cells but fell short of rendering them tumorigenic in vivo (37, 38). EZH2 promotes Ewing’s sarcoma growth. Among transcripts that were up-regulated in hMSC EWS-FLI-1 , we identified the gene encoding EZH2, a member of the polycomb group proteins (Supplementary data 2), which has recently been found to be expressed in ESFT (21). Together with EED and SUZ12, EZH2 forms the polycomb-repressive complex 2, believed to be a key regulator of embryonic development, stem cell renewal, and differentiation (39, 40). EZH2 is the catalytically active component of polycomb- repressive complex 2 and is believed to silence target genes by acting as a methyltransferase specific for Lys 27 of histone H3 and Lys 26 of histone H1 (reviewed in ref. 22). Overexpression of EZH2 has been shown to induce a bypass of the cellular senescence program in mouse embryonic fibroblasts and to prevent mouse hematopoietic stem cell exhaustion (41). Conversely, transient knockdown of EZH2 in primary human fibroblasts and a variety of transformed cells inhibited their proliferation in vitro (42). EZH2 expression has been proposed to be controlled by the Rb-E2F pathway and to be a downstream mediator of E2F-dependent proliferation (42). High EZH2 expression that has been observed in a broad range of tumors has thus far been attributed either to Rb loss or gene amplification (42). Its silencing function is speculated to target tumor suppressor genes (43), but the precise mechanism of its action on cell proliferation has thus far not been elucidated. Because of its role in stem cell maintenance and possibly tumor initiation (42, 44), we addressed the putative implication of EZH2 in ESFT pathogenesis. The EZH2 gene was found to be up- regulated in ESFT compared with normal tissues and neuroblastomas (21), and quantitative real-time PCR analysis confirmed EZH2 induction in hMSC EWS-FLI-1 as well as its dependence on EWS-FLI-1 DBD integrity (Fig. 4 A and data not shown). Expression of EZH2 has been reported to progressively decrease during serial passage of primary fibroblasts and hematopoietic stem cells cultured in vitro , resulting in activation of their senescence program (42). We therefore asked whether the elevated EZH2 expression observed in hMSC EWS-FLI-1 was the result of true up-regulation or mere maintenance of EZH2 transcripts at the level found in low-passage hMSCs. EZH2 expression levels in two different batches of empty vector–, DBDM-, and EWS-FLI-1– infected hMSCs after 20 doublings were compared by real-time PCR with those in parental wt hMSCs after 8 doublings. A 50% decrease in EZH2 expression was observed in cells infected with empty vector and the DBDM that had undergone 20 population doublings compared with parental wt cells that had undergone 8 doublings. By contrast, hMSC EWS-FLI-1 , after 20 doublings, showed a 6- to 7-fold induction of EZH2 expression compared with their 8 population doubling wt counterparts (Fig. 4 A ). This result clearly shows the ability of EWS-FLI-1 to induce EZH2 in hMSCs above the baseline level of their precursors that had undergone fewer population doublings, and is consistent with a possible role in both senescence prevention and stem cell maintenance in hMSCs. We next tested Ewing’s sarcoma samples and xenotransplants of Ewing’s sarcoma cell lines for EZH2 expression and found it to be elevated in both primary Ewing’s sarcoma and ESFT cell line–derived tumors grown in immunocompromised mice (Fig. 4 B ). To address the possible functional implication of EZH2 in Ewing’s sarcoma growth, we relied on a stable shRNA knockdown approach in ESFT cell lines using two distinct RNAi sequences (shRNA1 and shRNA2). Stable EZH2 shRNA expression in A673 and SK-N-MC Ewing’s sarcoma cell lines resulted in >70% reduction of its mRNA level (Fig. 4 C, left ) and a significant decrease in EZH2 protein expression as assessed by Western blot analysis (Fig. 4 C, right ). Reduction of EZH2 expression caused a marked decrease in proliferation of the two cell lines in vitro (Fig. 4 D ). Furthermore, injection of these cells into immunocompromised mice resulted in either no tumor development or strongly reduced tumor growth compared with cells expressing unrelated RNAi sequences (Fig. 5). To exclude possible off-target effects of the shRNA sequences, we repeated these experiments using two additional distinct shRNA sequences and obtained comparable results (data not shown). Based on the notion that EZH2 may repress p16 and possibly other cell cycle regulators (43), we tested the expression level of several cell cycle control genes in the EZH2-depleted cells by real- time PCR. Both of the ESFT cell lines used in our experiments have a nonfunctional p53 pathway (45). The A673 cell line also lacks the p16 INK4A -p14 ARF locus whereas SK-N-MC cells express an inactive Rb protein but retain expression of wt p16 INK4A -p14 ARF (45). Whether or not they were mutated, we did not record any significant change in the expression levels of p14 ARF , p15, p16 INK4A , p18, p21, and p53 following the EZH2 knockdown (data not shown). The observed reduction in proliferation and tumorigenicity can therefore not be explained by an effect on these cell cycle inhibitors and is most likely mediated by a mechanism that has yet to be elucidated. EWS-FLI-1 has thus far been found to induce an oncogenic stress type response in primary human cells, leading to cell cycle arrest, and a partial ESFT transcriptome in a variety of heterologous cell lines. Our present observations identify MSCs as the first primary human cell type that, while accurately recapitulating the ESFT gene expression profile, maintains viability and proliferation in response to EWS-FLI-1. EWS-FLI-1 induced marked rounding of hMSCs in vitro that accompanied robust expression changes of genes implicated in cell differentiation. Consistent with previous studies in tumor cells and immortalized fibroblasts, microarray analysis revealed that numerous genes induced in hMSC EWS-FLI-1 are implicated in neural crest development and neuronal differentiation. Among the most differentially expressed transcripts, we found NGFR (p75), which was strongly induced at both the transcription and protein levels, and which, in addition to playing a central role in neural develop- ment, is a key marker of neuroectodermal stem cells in both normal tissues and neural crest–derived tumors (46). Among the other genes that constitute part of the neuroectodermal profile of hMSC EWS-FLI-1 , SOX2 has been shown to maintain neural progenitor features (47). In contrast to observations in mouse mesenchymal progenitor cells, which were reported to lose osteogenic and adipocytic differentiation potential as a result of EWS-FLI-1 expression, hMSCs expressing EWS-FLI-1 retained at least some degree of trilineage differentiation plasticity. It is plausible that the early- stage neuroectodermal differentiation program induced by EWS- FLI-1 in hMSCs can still be overridden by the supraphysiologic conditions of the in vitro differentiation assays. Some of the genes that are implicated in neural differentiation have recently been suggested to play an active role in ESFT pathogenesis. Thus, NROB1 can promote tumorigenesis of Ewing’s sarcoma cell lines (7), whereas NKX2-2 may provide important functions at specific stages of ESFT development because its repression strongly reduced ESFT cell line tumorigenicity (10). It is noteworthy that both genes were not only strongly induced in hMSC EWS-FLI-1 but also remained dependent on EWS-FLI-1 expression in established and tumorigenic ESFT cell lines. These observations suggest that they play a role not only during the initial steps of tumor development but also at late stages of tumor growth. Importantly, several of the above genes have been found to be strong discriminators of ESFT, and their expression has been used as an argument that ESFT may be of neuroectodermal origin. However, the present experiments show that expression of genes implicated in neuronal differentiation and neural crest development can be induced by EWS-FLI-1 in the appropriate primary mesenchymal progenitor cell environment. Thus, a hMSC that can undergo partial neuroectodermal differentiation may constitute the origin of ESFT, suggesting that these tumors need not arise from a neuroectodermal precursor to explain their primitive neuroectodermal phenotype. Several of the genes observed to be up-regulated in hMSC EWS-FLI-1 and expressed in ESFT have been proposed to be functionally related to Ewings’ sarcoma development and behavior. Thus, ID2 , observed to be induced by EWS-FLI-1 in both hMSCs and mouse mesenchymal progenitor cells (20), may play an important role in promoting cell cycle entry by inhibiting Rb. Induction of fos that was detected as a result of EWS-FLI-1 introduction into hMSCs (Supplementary data 1C) may be of particular interest in light of recent evidence that expression of numerous candidate EWS-FLI-1 target genes requires cooperation ...
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Citations
... Poor differentiation is a feature of ES tumors, which might originate from different types of stem cells. The evidence indicates that ES may arise from either developing neural crest cells or mesenchymal stem cells [20,21,[31][32][33][34][35]. ES tumors likely contain a subpopulation of cells with stem cell features. ...
Retinoic acid (RA) regulates stemness and differentiation in human embryonic stem cells (ESCs). Ewing sarcoma (ES) is a pediatric tumor that may arise from the abnormal development of ESCs. Here we show that RA impairs the viability of SK-ES-1 ES cells and affects the cell cycle. Cells treated with RA showed increased levels of p21 and its encoding gene, CDKN1A. RA reduced mRNA and protein levels of SRY-box transcription factor 2 (SOX2) as well as mRNA levels of beta III Tubulin (TUBB3), whereas the levels of CD99 increased. Exposure to RA reduced the capability of SK-ES-1 to form tumorspheres with high expression of SOX2 and Nestin. Gene expression of CD99 and CDKN1A was reduced in ES tumors compared to non-tumoral tissue, whereas transcript levels of SOX2 were significantly higher in tumors. For NES and TUBB3, differences between tumors and control tissue did not reach statistical significance. Low expression of CD99 and NES, and high expression of SOX2, were significantly associated with a poorer patient prognosis indicated by shorter overall survival (OS). Our results indicate that RA may display rather complex modulatory effects on multiple target genes associated with the maintenance of stem cell’s features versus their differentiation, cell cycle regulation, and patient prognosis in ES.
... Given that gene expression levels in cancer cells can be indicative of an overlapping biology of normal tissues, we next examined the expression of the surface markers which are overexpressed in Ewing sarcoma in 7,862 normal tissues from 32 tissues of origin ( Figure S6B). Several genes, including ITM2A and FCGRT, both of which had been previously identified as EWS-FLI1 target genes, 64,65 were widely expressed across nearly all normal tissue, limiting their potential as therapeutic targets. In contrast, SLCO5A1, NPY5R, PCDH17, and CDH8 A C B D Figure 6. ...
... Importantly, several OFPs of LLPS scaffolds have been already shown to undergo LLPS, such as those of the FET family proteins (FUS, EWSR1 and TAF15) [83][84][85][86][87] and nucleoporins [77,[88][89][90] and some others, such as NONO-TFE3 [69], SS18-SSX [91] and BRD4-NUTM1 [92]. Most of these fusion products are primary drivers of cancer, i.e. they are potent oncogenes with the ability to drive the tumorigenic transformation of healthy cells by themselves [68,[93][94][95][96][97]. In their case, oncogenicity is mostly attributed to their ability to form condensates at non-native subcellular locations [54]. ...
Numerous cellular processes rely on biomolecular condensates formed through liquid-liquid phase separation (LLPS), thus, perturbations of LLPS underlie various diseases. We found that proteins initiating LLPS are frequently implicated in somatic cancers, even surpassing their involvement in neurodegeneration, where several such LLPS scaffolds are linked to disease emergence. Cancer-associated LLPS scaffolds are connected to all cancer hallmarks and tend to be oncogenes with dominant genetic effects currently lacking therapeutic options. Since most cancer-associated LLPS scaffolds exert their oncogenic effects as oncogenic fusion proteins (OFPs), we undertook a systematic analysis of known OFPs by assessing their module-level molecular functions. We identified both known and novel combinations of molecular functions that are specific to OFPs and thus have a high potential for driving tumorigenesis. Protein regions driving condensate formation show an increased association with DNA- or chromatin-binding domains of transcription regulators within OFPs, indicating a common molecular mechanism underlying several soft tissue sarcomas and hematopoietic malignancies where phase-separation-prone OFPs form abnormal, ectopic condensates along the DNA, and thereby dysregulate gene expression programs.
... In this context, it has been shown that overexpression of EWS-FLI1 in murine MSCs leads to their transformation and the formation of sarcoma once implanted in vivo, with characteristics (CD99 expression) and morphology similar to those of ES (Torchia et al., 2003;Castillero-Trejo et al., 2005;Riggi et al., 2005). Expression of EWS-FLI1 in human MSCs stimulates expression of genes involved in neuronal differentiation, but is not sufficient to induce a tumor in vivo, in contrast to experiments with murine MSCs (Riggi et al., 2008). In addition, a study of transcriptomic profiles showed that ES cells deficient in EWS-FLI1 displayed mesenchymal characteristics (Tirode et al., 2007). ...
In Europe, with an incidence of 7.5 cases per million, Ewing sarcoma (ES) is the second most common primary malignant bone tumor in children, adolescents and young adults, after osteosarcoma. Since the 1980s, conventional treatment has been based on the use of neoadjuvant and adjuvant chemotherapeutic agents combined with surgical resection of the tumor when possible. These treatments have increased the patient survival rate to 70% for localized forms, which drops drastically to less than 30% when patients are resistant to chemotherapy or when pulmonary metastases are present at diagnosis. However, the lack of improvement in these survival rates over the last decades points to the urgent need for new therapies. Genetically, ES is characterized by a chromosomal translocation between a member of the FET family and a member of the ETS family. In 85% of cases, the chromosomal translocation found is (11; 22) (q24; q12), between the EWS RNA-binding protein and the FLI1 transcription factor, leading to the EWS-FLI1 fusion protein. This chimeric protein acts as an oncogenic factor playing a crucial role in the development of ES. This review provides a non-exhaustive overview of ES from a clinical and biological point of view, describing its main clinical, cellular and molecular aspects.
... Cell lineage-restricted susceptibility to cancer is not limited to childhood brain tumours but likely dictates the formation of most childhood leukaemias and solid tumours [84][85][86][87][88][89][90] , as well as certain adult cancer types. For example, the BRAF V600E mutation occurs in melanomas in which the tumour cells express a neural crest-like transcriptome, suggesting that this developmental state is competent for transformation 91,92 . ...
Cancer has been a leading cause of death for decades. This dismal statistic has increased efforts to prevent the disease or to detect it early, when treatment is less invasive, relatively inexpensive and more likely to cure. But precisely how tissues are transformed continues to provoke controversy and debate, hindering cancer prevention and early intervention strategies. Various theories of cancer origins have emerged, including the suggestion that it is 'bad luck': the inevitable consequence of random mutations in proliferating stem cells. In this Review, we discuss the principal theories of cancer origins and the relative importance of the factors that underpin them. The body of available evidence suggests that developing and ageing tissues 'walk a tightrope', retaining adequate levels of cell plasticity to generate and maintain tissues while avoiding overstepping into transformation. Rather than viewing cancer as 'bad luck', understanding the complex choreography of cell intrinsic and extrinsic factors that characterize transformation holds promise to discover effective new ways to prevent, detect and stop cancer before it becomes incurable.
... For this purpose, we measured DisP-seq signals in EWS-FLI1 depletion experiments, where the loss of the fusion protein is known to result in widespread changes in chromatin 25 . This process involves not only the loss of active signals at EWS-FLI1 binding sites but also the reactivation of normal mesenchymal differentiation programs that are typical of mesenchymal precursors, the putative cells of origin of Ewing sarcoma 10,25,[35][36][37] . Mesenchymal differentiation in Ewing sarcoma cells with reduced levels of EWS-FLI1 has also been linked to increased migration, invasion and metastatic potential 23,24,38 . ...
Intrinsically disordered regions (IDRs) in DNA-associated proteins are known to influence gene regulation, but their distribution and cooperative functions in genome-wide regulatory programs remain poorly understood. Here we describe DisP-seq (disordered protein precipitation followed by DNA sequencing), an antibody-independent chemical precipitation assay that can simultaneously map endogenous DNA-associated disordered proteins genome-wide through a combination of biotinylated isoxazole precipitation and next-generation sequencing. DisP-seq profiles are composed of thousands of peaks that are associated with diverse chromatin states, are enriched for disordered transcription factors (TFs) and are often arranged in large lineage-specific clusters with high local concentrations of disordered proteins and different combinations of histone modifications linked to regulatory potential. We use DisP-seq to analyze cancer cells and reveal how disordered protein-associated islands enable IDR-dependent mechanisms that control the binding and function of disordered TFs, including oncogene-dependent sequestration of TFs through long-range interactions and the reactivation of differentiation pathways upon loss of oncogenic stimuli in Ewing sarcoma.
... S5, E and F). In addition, we also generated HiC profiles for primary MSCs, a model for the cell of origin of EwS (46,47). A/B compartment strength observed after EWS-FLI1 depletion in EwS cells resembled the values calculated for MSCs, suggesting the reestablishment of the compartmental segregation observed in these primary cells. ...
Cell fate transitions observed in embryonic development involve changes in three-dimensional genomic organization that provide proper lineage specification. Whether similar events occur within tumor cells and contribute to cancer evolution remains largely unexplored. We modeled this process in the pediatric cancer Ewing sarcoma and investigated high-resolution looping and large-scale nuclear conformation changes associated with the oncogenic fusion protein EWS-FLI1. We show that chromatin interactions in tumor cells are dominated by highly connected looping hubs centered on EWS-FLI1 binding sites, which directly control the activity of linked enhancers and promoters to establish oncogenic expression programs. Conversely, EWS-FLI1 depletion led to the disassembly of these looping networks and a widespread nuclear reorganization through the establishment of new looping patterns and large-scale compartment configuration matching those observed in mesenchymal stem cells, a candidate Ewing sarcoma progenitor. Our data demonstrate that major architectural features of nuclear organization in cancer cells can depend on single oncogenes and are readily reversed to reestablish latent differentiation programs.
... EWSR1::FLI1 is necessary to maintain Ewing sarcoma proliferation and survival and exerts an ability to transform human primary mesenchymal stem cells [55,56]. There are two common types of EWSR1::FLI1 fusions in patients: type 1 (fused by EWSR1 exon 7 with FLI1 exon 6) and type 2 (fused by EWSR1 exon 7 with FLI1 exon 5) fusions. ...
Simple Summary
Ewing sarcoma is a malignant pediatric bone cancer currently lacking targeted therapy. In the US there are ~200 patients diagnosed each year and relapse is associated with resistance to the standard-of-care chemotherapy. Thus, it remains an urgent unmet medical need to develop effective new cures for Ewing sarcoma. It is well-characterized that Ewing sarcoma is largely driven by unique gene fusions, with EWSR1-FLI1 being the most prevalent. In this review, we summarize up-to-date regulatory mechanisms for the onco-fusion protein EWSR1-FLI1 in Ewing sarcoma, including both post-transcriptional and post-translational modifications, to reveal knowledge gaps and propose potential new therapeutic directions.
Abstract
Ewing sarcoma is the second most common bone tumor in childhood and adolescence. Currently, first-line therapy includes multidrug chemotherapy with surgery and/or radiation. Although most patients initially respond to chemotherapy, recurrent tumors become treatment refractory. Pathologically, Ewing sarcoma consists of small round basophilic cells with prominent nuclei marked by expression of surface protein CD99. Genetically, Ewing sarcoma is driven by a fusion oncoprotein that results from one of a small number of chromosomal translocations composed of a FET gene and a gene encoding an ETS family transcription factor, with ~85% of tumors expressing the EWSR1::FLI1 fusion. EWSR1::FLI1 regulates transcription, splicing, genome instability and other cellular functions. Although a tumor-specific target, EWSR1::FLI1-targeted therapy has yet to be developed, largely due to insufficient understanding of EWSR1::FLI1 upstream and downstream signaling, and the challenges in targeting transcription factors with small molecules. In this review, we summarize the contemporary molecular understanding of Ewing sarcoma, and the post-transcriptional and post-translational regulatory mechanisms that control EWSR1::FLI1 function.
... The Ewing sarcoma fusion oncoprotein, EWS::FLI1, recruits the BAF chromatin remodeling complexes to activate its target genes [7]. Consistent with this, Slit2 silencing suppressed the expression of EWS::FLI1 target genes in Ewing sarcoma, including EZH2 [16,17], NKX2.2 [18], NPY1R [19], NGFR [20], NR0B1 [21], and PAPPA [22] ( Figure 6C). ...
Ewing sarcoma is a cancer of bone and soft tissue in children driven by EWS::ETS fusion, most commonly EWS::FLI1. Because current cytotoxic chemotherapies are not improving the survival of those with metastatic or recurrent Ewing sarcoma cases, there is a need for novel and more effective targeted therapies. While EWS::FLI1 is the major driver of Ewing sarcoma, EWS::FLI1 has been difficult to target. A promising alternative approach is to identify and target the molecular vulnerabilities created by EWS::FLI1.
Here we report that EWS::FLI1 induces the expression of Slit2, the ligand of Roundabout (Robo) receptors implicated in axon guidance and multiple other developmental processes. EWS::FLI1 binds to the Slit2 gene promoter and stimulates the expression of Slit2. Slit2 inactivates cdc42 and stabilizes the BAF chromatin remodeling complexes, enhancing EWS::FLI1 transcriptional output. Silencing of Slit2 strongly inhibited anchorage-dependent and anchorage-independent growth of Ewing sarcoma cells. Silencing of Slit2 receptors, Robo1 and Robo2, inhibited Ewing sarcoma growth as well. These results uncover a new role for Slit2 signaling in stimulating Ewing sarcoma growth and suggest that this pathway can be targeted therapeutically.
... The impact of cell of origin on tumor cell heterogeneity While the exact Ewing sarcoma cell or cells of origin is still an enigma, studies have shown that both neural crest stem cells (NCSCs) and mesenchymal stem cells (MSCs) tolerate EWS:: FLI1 expression and lead to increased Ewing-like gene expression and morphology (51)(52)(53)(54). Knockdown of EWS::FLI1 expression in Ewing sarcoma cells induces transcriptomes that closely resemble MSCs (18,50,55). ...
Accumulating evidence shows that despite clonal origins tumors eventually become complex communities comprised of phenotypically distinct cell subpopulations. This heterogeneity arises from both tumor cell intrinsic programs and signals from spatially and temporally dynamic microenvironments. While pediatric cancers usually lack the mutational burden of adult cancers, they still exhibit high levels of cellular heterogeneity that are largely mediated by epigenetic mechanisms. Ewing sarcomas are aggressive bone and soft tissue malignancies with peak incidence in adolescence and the prognosis for patients with relapsed and metastatic disease is dismal. Ewing sarcomas are driven by a single pathognomonic fusion between a FET protein and an ETS family transcription factor, the most common of which is EWS::FLI1. Despite sharing a single driver mutation, Ewing sarcoma cells demonstrate a high degree of transcriptional heterogeneity both between and within tumors. Recent studies have identified differential fusion protein activity as a key source of this heterogeneity which leads to profoundly different cellular phenotypes. Paradoxically, increased invasive and metastatic potential is associated with lower EWS::FLI1 activity. Here, we review what is currently understood about EWS::FLI1 activity, the cell autonomous and tumor microenvironmental factors that regulate it, and the downstream consequences of these activity states on tumor progression. We specifically highlight how transcription factor regulation, signaling pathway modulation, and the extracellular matrix intersect to create a complex network of tumor cell phenotypes. We propose that elucidation of the mechanisms by which these essential elements interact will enable the development of novel therapeutic approaches that are designed to target this complexity and ultimately improve patient outcomes.
