Luisa Cironi

EZH2 Is Essential for Glioblastoma Cancer Stem Cell Maintenance

Overexpression of the polycomb group protein enhancer of zeste homologue 2 (EZH2) occurs in diverse malignancies, including prostate cancer, breast cancer, and glioblastoma multiforme (GBM). Based on its ability to modulate transcription of key genes implicated in cell cycle control, DNA repair, and cell differentiation, EZH2 is believed to play a crucial role in tissue-specific stem cell maintenance and tumor development. Here, we show that targeted pharmacologic disruption of EZH2 by the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep), or its specific downregulation by short hairpin RNA (shRNA), strongly impairs GBM cancer stem cell (CSC) self-renewal in vitro and tumor-initiating capacity in vivo. Using genome-wide expression analysis of DZNep-treated GBM CSCs, we found the expression of c-myc, recently reported to be essential for GBM CSCs, to be strongly repressed upon EZH2 depletion. Specific shRNA-mediated downregulation of EZH2 in combination with chromatin immunoprecipitation experiments revealed that c-myc is a direct target of EZH2 in GBM CSCs. Taken together, our observations provide evidence that direct transcriptional regulation of c-myc by EZH2 may constitute a novel mechanism underlying GBM CSC maintenance and suggest that EZH2 may be a valuable new therapeutic target for GBM management.

Identification of Cancer Stem Cells in Ewing's Sarcoma

Cancer stem cells that display tumor-initiating properties have recently been identified in several distinct types of malignancies, holding promise for more effective therapeutic strategies. However, evidence of such cells in sarcomas, which include some of the most aggressive and therapy-resistant tumors, has not been shown to date. Here, we identify and characterize cancer stem cells in Ewings sarcoma family tumors (ESFT), a highly aggressive pediatric malignancy believed to be of mesenchymal stem cell (MSC) origin. Using magnetic bead cell separation of primary ESFT, we have isolated a subpopulation of CD133+ tumor cells that display the capacity to initiate and sustain tumor growth through serial transplantation in nonobese diabetic/severe combined immunodeficiency mice, re-establishing at each in vivo passage the parental tumor phenotype and hierarchical cell organization. Consistent with the plasticity of MSCs, in vitro differentiation assays showed that the CD133+ cell population retained the ability to differentiate along adipogenic, osteogenic, and chondrogenic lineages. Quantitative real-time PCR analysis of genes implicated in stem cell maintenance revealed that CD133+ ESFT cells express significantly higher levels of OCT4 and NANOG than their CD133- counterparts. Taken together, our observations provide the first identification of ESFT cancer stem cells and demonstration of their MSC properties, a critical step towards a better biological understanding and rational therapeutic targeting of these tumors.

EWS-FLI-1 Expression Triggers a Ewing's Sarcoma Initiation Program in Primary Human Mesenchymal Stem Cells

Ewings 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 cells, and the identity of permissive primary human cells that can support its expression and function has until now remained elusive. Here we show that expression of EWS-FLI-1 in human mesenchymal stem cells (hMSC) is not only stably maintained without inhibiting proliferation but also induces a gene expression profile bearing striking similarity to that of ESFT, including genes that are among the highest ESFT discriminators. Expression of EWS-FLI-1 in hMSCs may recapitulate the initial steps of Ewings sarcoma development, allowing identification of genes that play an important role early in its pathogenesis. Among relevant candidate transcripts induced by EWS-FLI-1 in hMSCs, we found the polycomb group gene EZH2, which we show to play a critical role in Ewings sarcoma growth. These observations are consistent with our recent findings using mouse mesenchymal progenitor cells and provide compelling evidence that hMSCs are candidate cells of origin of ESFT.

Expression of the FUS-CHOP Fusion Protein in Primary Mesenchymal Progenitor Cells Gives Rise to a Model of Myxoid Liposarcoma

A subset of sarcomas is associated with specific chromosomal translocations that give rise to fusion genes believed to participate in transformation and oncogenesis. Identification of the primary cell environment that provides permissiveness for the oncogenic potential of these fusion genes is essential to understand sarcoma pathogenesis. We have recently shown that expression of the EWS-FLI-1 fusion protein in primary mesenchymal progenitor cells (MPCs) suffices to develop Ewings sarcoma-like tumors in mice. Because most sarcomas bearing unique chromosomal translocations are believed to originate from common progenitor cells, and because MPCs populate most organs, we expressed the sarcoma-associated fusion proteins FUS/TLS-CHOP, EWS-ATF1, and SYT-SSX1 in MPCs and tested the tumorigenic potential of these cells in vivo. Whereas expression of EWS-ATF1 and SYT-SSX1 failed to transform MPCs, FUS-CHOP-expressing cells formed tumors resembling human myxoid liposarcoma. Transcription profile analysis of these tumors revealed induction of transcripts known to be associated with myxoid liposarcoma and novel candidate genes, including PDGFA, whose expression was confirmed in human tumor samples. MPC(FUS-CHOP) and the previously described MPC(EWS-FLI-1) tumors displayed distinct transcription profiles, consistent with the different target gene repertoires of their respective fusion proteins. Unexpectedly, a set of genes implicated in cell survival and adhesion displayed similar behavior in the two tumors, suggesting events that may be common to primary MPC transformation. Taken together, our observations suggest that expression of FUS-CHOP may be the initiating event in myxoid liposarcoma pathogenesis, and that MPCs may constitute one cell type from which these tumors originate.

Sarcomas: genetics, signalling, and cellular origins. Part 1: The fellowship of TET†

Sarcomas comprise some of the most aggressive solid tumours that, for the most part, respond poorly to chemo‐ and radiation therapy and are associated with a sombre prognosis when surgical removal cannot be performed or is incomplete. Partly because of their lower frequency, sarcomas have not been studied as intensively as carcinomas and haematopoietic malignancies, and the molecular mechanisms that underlie their pathogenesis are only beginning to be understood. Even more enigmatic is the identity of the primary cells from which these tumours originate. Over the past 25 years, however, several non‐random chromosomal translocations have been found to be associated with defined sarcomas. Each of these translocations generates a fusion gene believed to be directly related to the pathogenesis of the sarcoma in which it is expressed. The corresponding fusion proteins provide a unique tool not only to study the process of sarcoma development, but also to identify cells that are permissive for their putative oncogenic properties. This is the first of two reviews that cover the mechanisms whereby specific fusion/mutant gene products participate in sarcoma development and the cellular context that may provide the necessary permissiveness for their expression and oncogenicity. Part 1 of the review focuses on sarcomas that express fusion genes containing TET gene family products, including EWSR1, TLS/FUS, and TAFII68. Part 2 (J Pathol 2007; DOI: 10.1002/path.2008) summarizes our current understanding of the genetic and cellular origins of sarcomas expressing fusion genes exclusive of TET family members; it also covers soft tissue malignancies harbouring specific mutations in RTK‐encoding genes, the prototype of which are gastrointestinal stromal tumours (GIST). Copyright

IGF1 is a common target gene of Ewing's sarcoma fusion proteins in mesenchymal progenitor cells.

Background The EWS-FLI-1 fusion protein is associated with 85–90% of Ewings sarcoma family tumors (ESFT), the remaining 10–15% of cases expressing chimeric genes encoding EWS or FUS fused to one of several ets transcription factor family members, including ERG-1, FEV, ETV1 and ETV6. ESFT are dependent on insulin-like growth factor-1 (IGF-1) for growth and survival and recent evidence suggests that mesenchymal progenitor/stem cells constitute a candidate ESFT origin. Methodology/Principal Findings To address the functional relatedness between ESFT-associated fusion proteins, we compared mouse progenitor cell (MPC) permissiveness for EWS-FLI-1, EWS-ERG and FUS-ERG expression and assessed the corresponding expression profile changes. Whereas all MPC isolates tested could stably express EWS-FLI-1, only some sustained stable EWS-ERG expression and none could express FUS-ERG for more than 3–5 days. Only 14% and 4% of the total number of genes that were respectively induced and repressed in MPCs by the three fusion proteins were shared. However, all three fusion proteins, but neither FLI-1 nor ERG-1 alone, activated the IGF1 promoter and induced IGF1 expression. Conclusion/Significance Whereas expression of different ESFT-associated fusion proteins may require distinct cellular microenvironments and induce transcriptome changes of limited similarity, IGF1 induction may provide one common mechanism for their implication in ESFT pathogenesis.

Epigenetic Features of Human Mesenchymal Stem Cells Determine Their Permissiveness for Induction of Relevant Transcriptional Changes by SYT-SSX1

Background A characteristic SYT–SSX fusion gene resulting from the chromosomal translocation t(X;18)(p11;q11) is detectable in almost all synovial sarcomas, a malignant soft tissue tumor widely believed to originate from as yet unidentified pluripotent stem cells. The resulting fusion protein has no DNA binding motifs but possesses protein-protein interaction domains that are believed to mediate association with chromatin remodeling complexes. Despite recent advances in the identification of molecules that interact with SYT-SSX and with the corresponding wild type SYT and SSX proteins, the mechanisms whereby the SYT-SSX might contribute to neoplastic transformation remain unclear. Epigenetic deregulation has been suggested to be one possible mechanism. Methodology/Principal Findings We addressed the effect of SYT/SSX expression on the transcriptome of four independent isolates of primary human bone marrow mesenchymal stem cells (hMSC). We observed transcriptional changes similar to the gene expression signature of synovial sarcoma, principally involving genes whose regulation is linked to epigenetic factors, including imprinted genes, genes with transcription start sites within a CpG island and chromatin related genes. Single population analysis revealed hMSC isolate-specific transcriptional changes involving genes that are important for biological functions of stem cells as well as genes that are considered to be molecular markers of synovial sarcoma including IGF2, EPHRINS, and BCL2. Methylation status analysis of sequences at the H19/IGF2 imprinted locus indicated that distinct epigenetic features characterize hMSC populations and condition the transcriptional effects of SYT-SSX expression. Conclusions/Significance Our observations suggest that epigenetic features may define the cellular microenvironment in which SYT-SSX displays its functional effects.

Sarcomas: genetics, signalling, and cellular origins. Part 2: TET‐independent fusion proteins and receptor tyrosine kinase mutations

Although the mechanisms that underlie sarcoma development are still poorly understood, the identification of non‐random chromosomal translocations and receptor tyrosine kinase mutations associated with defined sarcoma types has provided new insight into the pathogenesis of these tumours. In Part 1 of the review (J Pathol 2007;213:4–20), we addressed sarcomas that express fusion genes containing TET gene family products. Part 2 of the review summarizes our current understanding of the implications of fusion genes that do not contain TET family members in sarcoma development, as well as that of specific mutations in genes encoding receptor tyrosine kinases (RTKs). The final section will serve as a summary of both reviews and will attempt to provide a synthesis of some of the emerging principles of sarcomagenesis. Copyright

The fusion protein SS18-SSX1 employs core Wnt pathway transcription factors to induce a partial Wnt signature in synovial sarcoma

Expression of the SS18/SYT-SSX fusion protein is believed to underlie the pathogenesis of synovial sarcoma (SS). Recent evidence suggests that deregulation of the Wnt pathway may play an important role in SS but the mechanisms whereby SS18-SSX might affect Wnt signaling remain to be elucidated. Here, we show that SS18/SSX tightly regulates the elevated expression of the key Wnt target AXIN2 in primary SS. SS18-SSX is shown to interact with TCF/LEF, TLE and HDAC but not β-catenin in vivo and to induce Wnt target gene expression by forming a complex containing promoter-bound TCF/LEF and HDAC but lacking β-catenin. Our observations provide a tumor-specific mechanistic basis for Wnt target gene induction in SS that can occur in the absence of Wnt ligand stimulation.