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Characterization of the EWS -FLI1 fusion protein in the tumorigenesis of Ewing's family of tumors

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Characterization of the EWS -FLI1 fusion protein in the tumorigenesis of Ewing's family of tumors

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Content CHARACTERIZATION OF THE EWS-FLI1 FUSION PROTEIN IN THE TUMORIGENESIS OF EWING’S FAMILY OF TUMORS Copyright 2004 by Siwen Hu A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (PATHOLOGY) December 2004 Siwen Hu Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3155425 Copyright 2004 by Hu, Siwen All rights reserved. INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. ® UMI UMI Microform 3155425 Copyright 2005 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Dedication F or m y parents, and m y husband, w ith love. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Acknowledgments I am most grateful for my mentor and committee chair Dr. Timothy J. Triche, for his support and trust, and for being a constant source o f encouragement and inspiration. Many thanks to my qualifying exam committee and dissertation committee members Dr. David Warburton Dr. Louis Dubeau Dr. David Hinton Dr. Alan Epstein for providing their expertise and helpful advice. I greatly appreciate the kind assistance, suggestions and inspirations from the members of The Triche lab (and office): Xian-Fang Liu, Lourdes Cruz, Dr. Jinsong Zhang, Dr. Chung Ho Shum, Nicole Tedeschi, Daniel Wai, Elai Davicioni The Wu lab: Dr. Lingtao Wu, Cheng Chen, Dr. Jiwei Wang The Anderson lab: Dr. Michael Anderson, Violette Shahbazian The Microarray Core at CHLA: Dr. Deborah Schofield, Betty Schaub, Sitara Waidyaratne, Xuan Chen The EM Core at CHLA: Dr. Hiro Shimada, Darkin Chan, Minerva Mongeotti The Clinical Pathology Lab at CHLA: Morgan Wu The Imaging Core at CHLA: Dr. George Mcnamara The Radiology Imaging Group at CHLA: Dr. Rex Moats, Dr. Mike Rosol, Maria Mouchess The Davis Group at California Institute of Technology: Dr. Mark E. Davis, Jeremy D. Heidel, Derek W. Bartlett And many many others........ iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table of Contents Dedication................................................................................................ii Acknowledgements............................................................................... iii List of Figures........................................................................................ vi List of Tables....................................................................................... viii Abstract...................................................................................................ix Introduction.............................................................................................1 Chapter I EWS-FLI1 fusion protein up-regulates critical genes in neural crest development and is responsible for the observed phenotype of Ewing’s family of tumors.....................................................................20 ABSTRACT................................................................................20 INTRODUCTION......................................................................22 RESULTS....................................................................................25 DISCUSSION.............................................................................47 EXPERIMENTAL PROCEDURES........................................ 56 Chapter 2 Secondary genetic alteration is required for EWS-FLI1 mediated oncogenesis............................................................................................ 64 INTRODUCTION......................................................................64 RESULTS....................................................................................67 DISCUSSION..............................................................................76 EXPERIMENTAL PROCEDURES........................................ 78 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 3 Investigating the potential of targeting EWS-FLI1 fusion gene as an adjuvant therapy for EFT patients..................................................... 80 INTRODUCTION.....................................................................80 RESULTS................................................................................... 86 DISCUSSION.............................................................................96 EXPERIMENTAL PROCEDURES........................................99 Chapter 4 Concluding remarks........................................................................... 103 Bibliography........................................................................................106 Appendix 1 Selective Usage of D Type Cyclins by Ewing’s Tumors and Rhabdomyosarcomas..........................................................................118 Appendix 2 Lack Of Interferon Response In Animals To Naked siRNAs 160 Appendix 3 Primers used in this study..................................................................191 V Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. List of Figures Figure 1. Schematic diagram of t(ll;22) associated with ES/PNET..................... 3 Figure 2. Structures of the EWS and FLU genes....................................................6 Figure 3. Domains of gene expression in the somites.............................................14 Figure 4. Potential Regulatory Networks Controlling Somitic Expression of MyoD and Myf-5............................................................................................................15 Figure 5. The TRex system.......................................................................................... 19 Figure 6. Microarray analysis revealed a global gene expression change and induction of EFT markers after E-F expression in RD cells................................. 26 Figure 7. Cultured RD-EF with E-F expression and xenograft tumors formed by E-F expressing RD showed the phenotype of typical EFTs..............................31 Figure 8. The appearance of an Ewing-like neuroectodermal phenotype is accompanied by a depletion of myogenic differentiation in induced RD-EF 33 Figure 9. IHC of xenografts.........................................................................................35 Figure 10. Comparison of up-regulated genes in RD-EF and highly expressed genes in EFT revealed important E-F target genes.................................................41 Figure 11. Canonical Wnt signaling is not active in EFT.....................................45 Figure 12. Gene regulation network initiated by EWS-FLI1................................53 Figure 13. EWS-FLI1 increases cyclin D1 but decreases cyclin D3 expression in RD cells....................................................................................................................... 68 Figure 14. EWS-FLI1 expression did not change the CDK4 associated D cyclins in RD cells..........................................................................................................70 Figure 15. EWS-FLI1 expression in RD cells induced a non-apoptotic G1 phase growth arrest...................................................................................................... 72 Figure 16. P16 protein level decreased after EWS-FLI1 expression in RD 75 Figure 17. Protein level of the Cip/Kip family of CKIs after expression of vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. EWS-FLI1 in RD-EF ceils 75 Figure 18. Conventional treatment of EFT...............................................................81 Figure 19. Mammalian cells have at least two pathways that compete for dsRNA..............................................................................................................................83 Figure 20. Structure of the key components of the delivery system.................... 84 Figure 21. In vitro study of the siRNA delivery to TC71 EFT cell line............... 87 Figure 22. Establishment of an EFT metastasis model...........................................90 Figure 23. Bioluminescent signal of the tumors decreased right after the fully formulated siLuc treatment.........................................................................................92 Figure 24. Effect of siEFBP2/CDP complex on the growth of metastasized EFTs.................................................................................................................................94 vii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. List of Tables Table 1. List of gene fusions involving EFT................................................................1 Table 2. The MyoD Family of Myogenic Regulators ..............................................13Table 3. Comparison of EFT, RMS and biphenotypic sarcomas......................... 16 Table 4.109 genes significantly up regulated in RD-EF and also highly expressed in EFT............................................................................................................38 viii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Abstract Ewing’s family of tumors (EFT) is consistently associated with an EWS- FLI1 translocation and a primitive neuroectodermal phenotype. Whether the translocation determines the phenotype, or vice versa, is debatable. Histogenesis and classification are therefore uncertain. To elucidate the role of EWS-FLI1 in the tumorigenesis and phenotype determination of EFT, we established a tetracycline- inducible E-F expression system in a rhabdomyosarcoma cell line RD (RD-EF). Expression of EWS-FLI1 in RD diminished the existing myogenesis and imposed an Ewing-like primitive neural crest phenotype, as evidenced by 1) Cell morphology changed after EWS-FLI1 expression, resembling cultured EFT cells. 2) Xenografts showed typical EFT features, distinct from tumors formed by parental RD. 3) Neuron-specific microtubule gene MAPT, parasympathetic marker CCK, and epithelial marker Keratin 18 were up regulated. Comparison of the up-regulated genes in RD-EF with the Ewing’s genes identified important EWS-FLI1 downstream genes, many involved in neural crest differentiation. These results were validated by real-time PCR analysis and RNA interference technology using siRNA targeting EWS-FLI1 breakpoint. The present study demonstrates that the primitive neuroectodermal phenotype of Ewing’s tumors is attributable to the EWS-FLI1 expression regardless of tissue of origin. The resultant phenotype resembles developing neural crest and simultaneously appears to limit terminal differentiation. In myogenic tissues, the result can be a primitive bi-phenotypic sarcoma with mixed myogenic and neural ix Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. phenotype. Our findings challenge traditional views of histogenesis and tumor origins. Finally, we established a highly reproducible mouse metastasis model for Ewing family of tumors. In vivo bioluminescent imaging system (Xenogen) allowed monitoring tumor growth over time in the same mouse. In addition, we showed that transferrin-conjugated cyclodextrin-mediated delivery of siRNA targeting EWS-FLI1 breakpoint resulted in transient growth inhibition of metastasized EFT tumors in mice. Cyclodextrin-based siRNA transfer system represents an attractive method to achieve maximal function of siRNA-based gene silencing in vivo. Our study showed that fully formulated siRNA targeting EWS-FLI1 has the potential to be developed into an adjuvant therapeutic tool for treatment of EFT patients and prevention of tumor metastasis. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Introduction 1. EFT harbor consistent tumor-specific translocations and the resulting fusion proteins (e.g. EWS-FLI1) play key roles in the tumor genesis by acting as aberrant transcription factors. Ewing’s family of tumors (EFT) includes classical Ewing's sarcoma (ES) of bone and soft tissues, peripheral primitive neuroectodermal tumors (pPNET), Askin tumor, and other less frequent variants. ES arises most often from the bones and is the second most common primary pediatric bone tumor. The most common sites are the lower extremity (45%), the pelvis (20%), upper extremity (13%), axial skeleton and ribs (13%), and face (2%). On the other hand, pPNET may arise in bone but are located most often in soft tissues. It is frequently found in the chest wall (Askin tumor), paraspinal tissues, abdominal wall, head and neck, and extremities (Grier, 1997). EFT mainly afflicts adolescents with an overwhelming predominance of Caucasion population (95%) and is slightly more common in males than females (1.5:1). Table 1. List of gene fusions involving EFT Translocation Fusion Incidence EWS-FLI1 90-95% t(ll;22)(q24;ql2) Type 1 65% Type 2 25% Other types 10% t(21;22)(q22;ql2) EWS-ERG 5-10% t(7;22)(q22;ql2) EWS-ETV1 Rare t(17;22)(ql2;ql2) EWS-E1A Rare t(2;22)(q33;ql2) EWS-FEV Rare 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The various members of EFT were grouped into one tumor entity because of consistent tumor-specific translocations, which is common among mesenchymal tumors. In more than 95% of EFT cases, there is a characteristic chromosomal translocation t (11 ;22)(q24;q 12), which juxtaposes the proximal region of the ubiquitously expressed EWS gene on chromosome 22 to the distal region of FLU, a member of the ETS family of transcription factors (Figure 1) (Delattre et al., 1992; Zucman et al., 1992). Another four translocations have also been reported in EFT with similar fusion of the EWS gene with four different ETS members (Table 1). All of the fusion proteins contain the N-terminal transactivation domain of EWS and the C terminal domain of the ETS members, including the intact ETS consensus DNA binding domain which recognize a common purine-rich core motif of 5’-GGAA-3’ (Figure 1). These EWS-ETS translocations appear to be specific to EFTs and play key roles in tumor genesis by acting as aberrant transcription factors. They are important for tumor diagnosis, classification and micrometastasis detection, and are potential therapeutic targets. Secondary molecular alterations, including mutations of cell cycle regulatory genes, are not tumor-specific but are related to tumor progression and may have prognostic value. We focus on the EWS-FLI1 fusion since it is most frequently identified. 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 1. Schematic diagram of t (11;22) associated with ES/PNET (A) and the chimeric EWS-FLI1 protein (B). Adapted from Enrique de Alava, et al., 2000. B 11 1* $ 4 5 1 U as it ^ 15 P 11 11 n * 1 CHR 22 CHR 11 EW S(22q12} N H » i E W S -F L I1 t(11;22)der22 < » H 1 * 1 * *t n * * * « d»r 22 d«r 11 1(11 ;22)(q24;q12) WTl>«»g W N A B P M tiM M T t SPilCi triD W H O C O O H COOH FU 1 (11q24) N^f COOH Flil {Friend Leukemia Integration 1) is located on chromosome 11 and belongs to the ETS family of transcription factors (Graves and Petersen, 1998). They bind to the promoters or enhancer elements of target genes and act as transcriptional activators or repressors. The ETS members share a specific ETS DNA binding 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. domain at their C termini and form heteromeric complexes with other nuclear proteins at the target promoter. So the binding specificity is determined by both DNA-protein and protein-protein interactions. The N terminus of Flil functions as a transactivation domain although the C terminus has also been shown to have some transactivation potential (Rao et al., 1993). The expression of FLI1 is tightly regulated and lineage-restricted. During embryologic development Flil is found in neural crest and mesodermal cells (Mager et al., 1998) as well as in vascular and blood cell precursors. In adults Flil is predominantly expressed in hematopoietic cells (Brown et al., 2000; Melet et al., 1996). FLIl-knockout in mice is embryonically lethal because of hematopoiesis and vascular development defects (Truong and Ben-David, 2000). EWS (EW ing’ s Sarcoma) is a ubiquitously and highly expressed protein with RNA binding capability, which may function as an adaptor protein linking transcription with mRNA processing. The EWS gene contains 17 exons spanning about 40kb on chromosome 22. The N terminal domain of EWS shares distant homology with C-terminal of eukaryotic RNA Polymerase II (Ohno et al., 1994) and functions as strong transactivation domains. The Fusion of EWS with different non- ETS family translocation partners is involved in several other types of sarcomas (Sorensen and Triche, 1996). The EWS protein belongs to a TET family, which also includes TLS/FUS and TAFII68. This family of proteins associate with TFIID and RNA polymerase II subunits and may participate in the regulation of RNA transcription and processing (Bertolotti et al., 1998; Burd and Dreyfuss, 1994). 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The structure of EWS-FLI1 is heterogeneous. Eighteen possible types of in frame EWS-FLI1 chimeric transcripts resulting from the t(l 1 ;22)(q24;ql2) have been observed in vivo with considerable combinatorial diversity of junctions, reflecting genomic breaks in one of four EWS introns and one of six FLI1 introns (Figure 2) (Zucman et al., 1993). Among them, the type 1 (fusion of EWS exon 7 to FLI1 exon 6) and type 2 (fusion of EWS exon 7 to FLI1 exon 5) fusions are most common, accounting for about 85-90% of EWS-FLI1. Different forms of EWS-FLI1 have different transactivating function. For example, inclusion of a variable portion of the FLI1 amino-terminal activation domain, or the IQ domain encoded EWS exon 8, enhances transactivation. The consequence of these functional differences is differences in the proliferative rate and clinical course of Ewing's sarcoma, i.g. type 1 EWS-FLI1 fusion transcript, the most common type, is associated with a lower proliferative rate and a favorable prognosis (15% vs. 24% for non-type 1 cases). Patients with ES and EWS-FLI1 transcripts other than type 1 have a poor prognosis, i.e. positive blood samples, tumor progression and rapid death from disease, regardless of stage, tumor location or age(de Alava et al., 1998a). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2. Structures of the EWS and FLU genes with arrowheads indicating the naturally occurring fusion sites in the chimeric transcripts resulting from the t(ll;22)(q24;ql2) with considerable combinatorial diversity of junctions. Adapted from Sandberg and Bridge, 2000 transactivating domain RNA binding CM/C m m wW fusion sites cDNA in s e rts pEF7-4 (type 2) pEF7-5 pEF7*7 pEF7-9 transactivating DNA CTA domain BD domain « ----------I I 1 I ' I 3 = 3 FU1 fusion sites (type 1) pEF7-6 3M EO .IJ pEF7-8 pEF10*6 9 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The chimeric EWS-Flil protein appears to act as an aberrant transcription factor. It localizes to the nucleus of EFT cells (Bailly et al., 1994) and can bind to DNA via the ETS domain with similar DNA binding specificity and affinity as FLU (Mao et al., 1994). In a reporter model system, the N terminal domain of EWS, which has remained in the fusion protein, has been shown to be a more powerful transactivation domain than the N terminal domain of wild type FLI1 (May et al., 1993; Ohno et al., 1993). The EWS promoter, strongly and broadly activated, controls the fusion gene transcription, therefore, the EWS-FLI1 protein is expressed at unrestricted high level in the cells that normally don’t express FLI1, resulting in the deregulation of the expression of downstream target genes, largely unknown but critical to cell proliferation and differentiation. Thus far the reported EWS-FLI1 direct and indirect targets include: PDGF-C (Zwemer and May, 2001), p57KIP2 and MYC(Dauphinot et al., 2001), TGFBR2 (Hahm et al., 1999), ID2 (Fukuma et al., 2003; Nishimori et al., 2002) and CYCLIN D1 (Matsumoto et al., 2001), etc. In addition, it has been reported that modulation of the MAP kinase pathway by EWS- FLI1 is probably important in the tumorigeneis of EFT (Silvany et al., 2000). 2. Ewing's family of tumors has limited parasympathetic neuroectodermal features although the cell of origin is still unknown. EFT, along with rhabdomyosarcoma (RMS), neuroblastoma (NB) and Non- Hodgkin’s lymphoma, belongs to small round blue cell tumors of childhood (SRBCT), a group of very primitive childhood tumors with similar histological appearance as poorly differentiated “sheets of cells” with small round blue nuclei. 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The correct clinical diagnosis of SRBCT has been difficult by light microscopy alone because of the lack of characteristic morphologic appearances. Indeed, although James Ewing (Ewing, 1921) and Arthur Purdy Stout (Stout, tti 1918) described Ewing’s sarcoma and pPNET, respectively, in the early 20 century, the histogenesis of EFT is still debatable. A neural crest progenitor has been suggested (Dehner, 1993; Kovar, 1998) because the tumors exhibit variable expression of neuronal markers (such as catachol acetylcholine transferase and neuron-specific enolase), ultrastructural features (Homer-Wright rosettes, neural processes, neurosecretory granules) (Hartman et al., 1991), and the ability of EFT cell lines to express neural associated proteins and form primitive dendrites in response to differentiating agents in vitro (Cavazzana et al., 1987; Noguera et al., 1992). However, EFT can also exhibit some epithelial and mesenchymal characteristics and can arise in organs not developed from the neural crest (eg, the kidney). In contrast to NB, another SRBCT that has a sympathetic neural crest origin, most EFT express the MIC2 gene at extremely high levels, whose product is a glycoprotein (CD99) located on the cell surface and involved in cell adhesion (Ambros et al., 1991; Fellinger et al., 1991). Membrane MIC2 expression is a sensitive but not specific marker for the EFT diagnosis(Weidner and Tjoe, 1994). In addition, nearly half of primary NBs and all NB cell lines show N-MYC amplification, however, ES and pPNET always lack N-MYC expression. Instead, most EFTs over-expresses C-MYC. Therefore, EFT has been considered to derive 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. from a postganglionic parasympathetic primordial cells because of the following evidence: 1) EFT tumors synthesize acetylcholine transferase and do not synthesize adrenergic precursors. EFT cell lines are capable of acetylcholine synthesis too, although they lack an organized mechanism for acetylcholine release (O'Regan et al., 1995). 2) EFT cell lines have receptors for neuropeptide Y (a parasympathetic regulatory polypeptide (van Valen et al., 1992)), and receptors for 13-adrenergic and dopamine D-l (van Valen and Keck, 1988). 3) The mRNA and peptides of the neurotransmitter cholecystokinin (CCK) are strongly expressed by EFT (Friedman et al., 1992). Neuroblastoma and osteosarcoma (OS), on the other hand, don’t express this gene. 3. EWS-FLI1 fusion gene functions as a dominant oncogene. However, EWS- FLI1 alone is not sufficient to transform most cell types. Previous studies suggest that the EWS-FLI1 fusion protein is an important factor in tumor genesis of EFT and functions as a dominant oncogene, i.e. promoting the cell proliferation. Indeed, NIH 3T3 cells can be transformed by forced EWS- FLI1 expression and can acquire the capability of anchorage-independent growth and tumor formation in immuno-deficient mice (May et al., 1993; Thompson et al., 1999). Interestingly, xenograft tumors ffomed by EWS-FLI1 transformed NIH 3T3 cells present a small round cell morphology instead of the typical spindle-shaped transformed fibroblasts (Teitell et al., 1999). Wild type Flil does not have this capability, which suggests that a permissive cellular context is important for the function of this chimeric fusion gene and other cellular proteins may modulate the function of EWS-FLI1. Both EWS and Flil domain are required for this 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. transformation. When EWS is replaced by other strong transactivation domains (VP 16, ETS2, or FUS), the new fusion constructs can also render NIH 3T3 cells anchorage independent (Lessnick et al., 1995). This implies that the primary role of EWS in the fusion protein is transcription activation. Conversely, abrogation of EWS-FLI1 expression in EFT cell lines resulted in decreased cell proliferation and loss of tumorigenicity (Lambert et al., 2000; Ouchida et al., 1995; Tanaka et al., 1997). However, it was later discovered that simple transfection of the E-F gene was generally lethal, and certainly did not accelerate cell proliferation (Deneen and Denny, 2001; Lessnick et al., 2002). This in fact has been true of most such chimeric oncogenes when simply transfected into normal or tumor cell backgrounds, and indicates that the fusion protein by itself may not be sufficient for transformation and secondary genetic alterations are required. 3. EWS-FLI1 inhibits existing tissue-associated differentiation, however, E-F transformed NIH 3T3 cells acquire a characteristic small round cell morphology and some neural features. The occurrence of EWS-FLI1 (EWS-ETS) translocation(s) has enabled grouping of a spectrum of seemingly unrelated tumors with various degrees of restricted neuroectodermal differentiation into one unified family: from typical undifferentiated Ewing’s sarcoma to poorly differentiated atypical Ewing’s sarcoma to differentiated peripheral primitive neuroectodermal tumor (pPNET) The consistency and tumor specificity of the translocations in EFT implies a close relation between expression of chimeric oncoproteins and the primitive neural 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. phenotypes of EFT. However, it is unclear whether EWS-FLI1 blocks differentiation in neuroectodermally derived cells with an otherwise greater capacity for neural development, or it contributes to the neural phenotype in a primitive pluri-potential stem cell. Recent studies showed that EWS-FFLI1 appears to inhibit tissue-specific differentiation. Forced E-F expression inhibited osteogenic and adipogenic differentiation in marrow stromal cells (Torchia et al., 2003), and myogenic differentiation in C2C12 cells. Interestingly, tumors formed by E-F-transformed NIH3T3 cells (an immortalized murine fibroblast line, the only cell line that can be transformed by EWS-FLI1 so far), acquired a certain degree of neural features and a small round cell morphology, which is typical of EFT, but distinct from fibrosarcomas (Teitell et al., 1999). These results suggest a possible role of EWS- FLI1 in inhibiting existing tissue-associated differentiation but promoting a neurectodermal differentiation program in these tumors. 4. RMS, another member of the SRBCT group, arises from myogenic progenitors and expresses the most important myogenic regulator MyoD. Rhabdomyosarcomas (RMS) is another member of the SRBCTs. They show features of muscle differentiation (cross-striations, muscle specific protein expression, etc.) and histologically resemble embryonic skeletal myoblasts (Scrable et al., 1989). RMS also expresses the MyoD family of myogenic regulators, including MyoD (Myf3), Myf5 (Myf5), myogenin (Myf4) and MRF4 (Myf6) [mice (human)] (Tonin et al., 1991). The MyoD family belongs to a bHLH (basic Helix-Loop-Helix) family of transcription factors and is capable of committing the precursor cells to the skeletal 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. muscle lineage (Buckingham, 1992; Buckingham, 1994). They heterodimerize with one of the ubiquitous E proteins (El 2, E47) and activate muscle specific genes by binding to the E box (consensus DNA sequence CANNTG) in the target promoters or enhancers. The four MyoD family members can be divided into two classes, determination factors and differentiation factors (Olson and Klein, 1994). MyoD and Myf5 are expressed early in myogenesis and specify the muscle lineage (determination factors) (Comelison and Wold, 1997; Smith et al., 1994). They share functional redundancy since MyoD or Myf5 knockout mice can form essentially normal skeletal muscle but mice with double MyoD/Myf5 knockout have no skeletal muscle (Rudnicki et al., 1993). However, MyoD is critically required in adult muscle regeneration (Megeney et al., 1996). In MyoD mutant mice, the adult muscle stem cells, also known as “satellite cells”, show deficiency in proliferation during muscle regeneration. Myf5 cannot substitute for this function. Myogenin and MRF4 are expressed at late stage of muscle development and activate the muscle cell differentiation program (differentiation factors). Myogenin mutant mice show normal MyoD and Myf5 expression and correctly specified myoblasts, but these myoblasts won’t differentiate and fuse into myofibers (Molkentin and Olson, 1996). 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. able 2. The MyoD Family of Myogenic Regulators (Knock-out Mice Study) Genotype Phenotype Conclusions MyoD-/- • Normal skeletal muscle at birth • Deficiency in adult muscle regeneration 1. Determination factors 2. Specify the muscle lineage 3. Share redundancy during embryogenesis 4. MyoD is crucial in adult muscle regeneration MyfS-/- • Normal skeletal muscle MyoD-/- AND MyfS-/- • Complete loss of skeletal muscle Myogenin-/- OR MRF4-/- • Normal MyoD and Myf5 expression • Correctly specified myoblasts but won’t differentiate and fuse into myofibers 1. Differentiation factors 2. Activate the muscle cell differentiation program In addition to the MyoD family of myogenic factors, Pax3 and Pax7 are also important upstream myogenic regulators. Pax3 and Pax7 belong to a paired-type homeobox (PAX) family of transcription factors. They are transiently expressed in tissues destined to become neurogenic and myogenic and play important roles in regulating embryonic development (Mansouri et al., 1999; Mansouri et al., 1996). During somitic myogenesis, the combination of Wntl and Shh (sonic hedgehog) signaling from the surrounding neural tube and notochord induces Pax3 and Pax7 expression in the somite, where all skeletal muscles arise except some head muscles Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Yun and Wold, 1996) (Figure 3). PAX3 functions upstream of MyoD and Myf5, and commits the multi-potent somitic cells into the muscle differentiation program (Maroto et al., 1997; Tajbakhsh et al., 1997). Pax3 is found to be a potent MyoD and Myf5 activator and forced expression of Pax3 can initiate muscle differentiation in the mesoderm and neural tube without inducing tissues (Figure 4). Pax7 is required for the specification of the adult satellite cells (Seale et al., 2000). There is a complete absence of satellite cells in Pax7 knockout mice. Figure 3. Domains of gene expression in the somites (a transverse section), ec, surface ectoderm; lb, limb bud; der, dermomyotome; my, myotome; scl, sclerotome; nt, neural tube; no, notochord. Adapted from Alan Rawls, et al., 1997. ec epaxial ■ Pax-3 □ Pax-7 f l j M yogenic bHLH D orsal i Medial 4 ----- ► Lateral Ventral 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 4. Potential Regulatory Networks Controlling Somitic Expression of MyoD and Myf-5. Adapted from Miguel Maroto, et al., 1997 Ectoderm Dorsal Neural tube' — ► Pax-3 (& Pax-7?)' Notochord/ Floor plate' c-met Migration of somitic cells into limb Activation of myogenin and muscle gene expression Ectoderm 5. A group of biphenotypic tumors contain the same EWS-FLI1 or EWS-ERG fusions as EFT and manifest a lesser degree of myogenic differentiation than RMS with no translocations, while displaying some neural features. RMS seems to be distinct from Ewing’s sarcoma since they are thought to arise from different precursor cells. However, the existence of so-called biphenotypic tumors makes this distinction obscure (de Alava et al., 1998b; Sorensen et al., 1995; Tan et al., 2001; Thomer et al., 1996). Sorensen et al. first reported a group of soft tissue sarcomas that were originally diagnosed as primitive RMS (Sorensen et al., 1995). Histologically these biphenotypic tumors are poorly differentiated rhabdomyoblasts separated by fibrous septae. However, immunostaining and ultrastructural study revealed evidence of both myogenic and neurogenic features, and the tumor expresses muscle-specific antigens as well as neural-associated genes. It is believed that about 5% of the diagnosed RMS tumors belong to this category 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (unpublished IRS IV data). Most interestingly, all of the tumor cells tested harbor the t(l 1;22) translocation identical to those of Ewing’s family of tumors, and EWS-Flil is expressed in these tumors. Myogenic regulatory factors were detected by Northern analysis. These tumors still express Myf5, however, MyoD expression is absent in all of the biphenotypic tumors tested. From our group’s previous study and one published report, Pax3 and Pax7 are moderately to highly expressed in EFTs, RMSs, as well as biphenotypic sarcomas (Bader et al., 1989; Tast, 1999), corresponding to their normal expression pattern during embryogenesis. Pax3 is a strong MyoD activator and ectopic Pax3 expression in dorsal neural tube has been shown to be capable of inducing expression of myogenic bHLH transcription factors, including MyoD (Maroto et al., 1997). However, MyoD is not expressed in EFTs and biphenotypic sarcomas, where Pax3 is expressed (Table 3). Table 3. Comparison of EFT, RMS and biphenotypic sarcomas m v i i l V Biphe. Sarc. V V - V V RMS — -y j V V V 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6. Hypothesis and objective of this study As an active oncogene, EWS-FLI1 has been extensively studied with respect to its ability to promote tumor cell proliferation and inhibit apoptosis (Arvand and Denny, 2001; de Alava and Gerald, 2000). However, the possible role of EWS-FLI1 in differentiation is still an untouched field. The fact that this translocation is tumor- specific (only in EFTs) and very consistent (identified in more than 85% of the patients) indicates a close relation between EWS-FLI1 expression and the tumor’s phenotype. There are two possibilities: (1) EWS-FLI1 fusion happens in a more determined neural cell lineage and inhibits the existing differentiation program while promoting cell proliferation. (2) Alternatively, the fusion happens in a multi-potent undifferentiated precursor cell and subsequently influences the cell development and imposes the observed phenotype on the tumor cells. We favor the latter because of the following evidence: Ewing family of tumors represents a continnum of tumors with various degree of primitive neuroectodermal differentiation. EWS-FLI1 transformed NIH 3T3 cells acquired a small round cell morphology typical of Ewing’s tumors and distinct from the spindle-shaped transformed fibroblasts. Biphenotypic sarcomas with EWS-FLI1 translocation show a more primitive morphology than that of the regular RMS without any translocation, and the tumor cells present neurogenic features in addition to myogenic features. Since EWS-FLI1 is a potent chimeric transcription factor, it may dictate the phenotype of Ewing’s family of tumors by deregulating expression of genes related to terminal differentiation. 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In this study, we performed a comprehensive study investigating the effect on cell differentiation and proliferation, by controlled expression of EWS-FLI1 fusion gene (The TRex system, Figure 5) in an embryonal rhabdomyosarcoma cell background. We also studied the feasibility of silencing EWS-FLI1 in vivo using RNA interference technology as an adjuvant therapy for EFT patients. 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5. The TRex system. A. Diagram of the two plasmids, pcDNA6/TR and pcDNA4/To/EF B. Mechanism of the TRex system. pcDNA6/TR 6662 bp TATA pcDNA4/TO 5078 bp f'A IA ^ Expression Derepressed Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 1 EWS-FLIl fusion protein up-regulates critical genes in neural crest development and is responsible for the observed phenotype of Ewing’s family of tumors Abstract Tumor-specific translocations are common in tumors of mesenchymal origin. Whether the translocation determines the phenotype, or vice versa, is debatable. Ewing’s “tumors” are consistently associated with an EWS-FLIl translocation and a primitive neural phenotype. Histogenesis and classification are therefore uncertain. To test whether EWS-FLIl (E-F) fusion protein expression is responsible for the primitive neuroectodermal phenotype of Ewing’s family tumors (EFTs), we established a tet-inducible E-F expression system in a rhabdomyosarcoma cell line RD (RD-EF). Cell morphology changed after E-F expression, resembling cultured EFT cells. Xenografts showed typical EFT features, distinct from tumors formed by parental RD. Neuron-specific microtubule gene MAPT, parasympathetic marker CCK, and epithelial marker Keratinl8 were up-regulated. Conversely, myogenesis was diminished. Comparison of the up-regulated genes in RD-EF with the Ewing’s genes identified important E-F downstream genes, many involved in neural crest differentiation. These results were validated by real-time PCR analysis and RNA interference technology using siRNA targeting E-F breakpoint. The present study demonstrates that the neural phenotype of Ewing’s tumors is attributable to the E-F 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. expression. The resultant phenotype resembles developing neural crest and simultaneously appears to limit terminal differentiation. Such tumors have a limited neural phenotype regardless of tissue of origin. In myogenic tissues, the result can be a primitive bi-phenotypic sarcoma with mixed myogenic and neural phenotype. These findings challenge traditional views of histogenesis and tumor origins. 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Introduction Mesenchymal tumors often harbor characteristic chromosome translocations (Rabbitts, 1994). The consistency and tumor-specificity of these translocations imply a close relation between the fusion proteins as a result of the translocations, and certain tumor phenotypes. One possible explanation is that a given translocation can only occur in a certain determined cell lineage where the right cellular background exists, to tolerate and cooperate with the fusion protein. Alternatively, distinct fusions may occur in common multi-potent undifferentiated precursor cells and influence cell development, subsequently driving the cells towards different phenotypes or lineages. A good model to investigate these possibilities is the Ewing’s family of tumors (EFT), a group of poorly differentiated pediatric and young adult cancers of bone and soft tissue, which harbors characteristic translocations in virtually all cases. These translocations fuse a heretofore unknown gene, termed EWS, on chromosome 22, with a member of the ETS family of developmentally regulated genes, most commonly FLI1 on chromosome 1 l(Delattre et al., 1992). These fusion proteins function as aberrant transcription factors. Numerous investigations have now documented the near universal (if not universal) association of one of these translocations with the tumor. The occurrence of EWS-FLIl (EWS-ETS) translocation(s) has enabled grouping of a spectrum of seemingly unrelated tumors with various degrees of neuroectodermal differentiation into one family: from typical undifferentiated Ewing’s sarcoma to poorly differentiated atypical Ewing’s sarcoma to differentiated 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. peripheral primitive neuroectodermal tumor (pPNET). The cell lineage that EFT originates from is still somewhat enigmatic. However, a parasympathetic neural crest origin has been suggested because some of the tumors express limited degree of neural markers (e.g. cholecystokinin (CCK) (Thiele, 1991)) and they can be induced to undergo neural differentiation by various differentiating agents (Cavazzana et al., 1987; Noguera et al., 1992). EWS-FLIl (E-F) has been considered a traditional “oncogene”, i.e. promoting the proliferation and blocking the differentiation of a committed neural crest precursor cell. Indeed early experiments that down-regulated expression of the chimeric gene resulted in diminished proliferation (Ouchida et al., 1995). However, it was later discovered that simple transfection of the E-F gene was generally lethal, and certainly did not accelerate cell proliferation (Deneen and Denny, 2001; Lessnick et al., 2002). This in fact has been true of most such chimeric oncogenes when simply transfected into normal or tumor cell backgrounds, and indicates that secondary genetic alterations are required for E-F mediated transformation. In contrast to its role in oncogenesis, E-F appears to inhibit tissue-specific differentiation. Forced E-F expression inhibited osteogenic and adipogenic differentiation in marrow stromal cells (Torchia et al., 2003), and myogenic differentiation in C2C12 cells. Interestingly, tumors formed by E-F-transformed NIH3T3 cells (an immortalized murine fibroblast line, the only cell line that can be transformed by EWS-FLIl so far), acquired a certain degree of neural features and a small round cell morphology, which is typical of EFT, but distinct from 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. fibrosarcomas (Teitell et al., 1999). This suggests a possible role of EWS-FLIl in inhibiting tissue-associated differentiation but promoting a neurectodermal differentiation program in these tumors. Further evidence is a group of biphenotypic soft tissue sarcomas (about 5% of the diagnosed rhabdomyosarcoma (RMS), unpublished IRS IV data). They contain the same EWS-FLIl or EWS-ERG fusions, and manifest a lesser degree of myogenic differentiation than RMS with no translocations while displaying some neural features (Sorensen et al., 1995; Tan et al., 2001; Thomer et al., 1996). In this study, we established a tetracycline (Tet) regulated E-F expression model in RD, an embryonal RMS cell line with marked myogenic differentiation, to test the hypothesis that F-F fusion protein is responsible for the observed primitive neuroectodermal phenotype of EFT, by the regulation of genes involved in cell proliferation and differentiation. 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Results Establishment of the RD-EF model system Rhabdomyogenesis has repeatedly been identified to co-exist with the phenotype of EFT (Sorensen et al., 1995; Tan et al., 2001; Thomer et al., 1996). Therefore, we expressed the E-F fusion gene in RD cells by using aTet-inducible system (TREx, Invitrogen), which allowed us to identify expression levels that are non-lethal but associated with profound effects on patterns of gene expression and cell differentiation. Several stable clones with high inducibility and low leakage were selected. Among them, RD-EF01 was used for the subsequent experiments because Tet treatment led to strong induction of E-F in this clone. E-F RNA could be detected as early as 3h by real-time RT-PCR (Fig 6A). Western blotting analysis revealed a rapid induction of E-F proteins as early as 6h and reached the peak level at 36h (Fig 6B). Fluorescent microscopic study showed that induced E-F was localized to the nucleus (Fig 6C). Since E-F functions as an aberrant transcription factor, to investigate the molecular consequences of EWS-FLIl expression in RD cells, we undertook a transcriptome-wide gene expression analysis using Affymetrix HU95av2 gene chips, at time 0, 6,12,18, 24 and 36 hours after Tet or EtOH treatment (2-6 biological replicates at each time point, Figure 6D). An additional set of RNA was harvested at time 0, 6, 9,18,24, and 36 hours for confirmatory quantitative RT-PCR using 3 actin as an internal control. We also compared the microarray data of the model system 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. with a pool of data from 83 tumor samples, including 16 EFT, 36 RMS, 20 osteosarcomas (OS) and 10 neuroblastomas (NB) (Fig 6F). Figure 6. Microarray analysis revealed a global gene expression change and induction of EFT markers after E-F expression in RD cells A. Real time PCR analyses of RD-EF cells after T (red line) or E (green line) induction. B. Western blot analyses of RD-EF cells after T (tetracycline) or E (ethanol) induction. Upper and lower panel represent the same blots probed with antibodies against Flil or Actin. C. E-F cellular localization in RD-EF cells was visualized by immuno-fluorescent microscopy. TC32 cells stained by a monoclonal antibody specific to human Fli- 1 (green signal). Nuclei were counterstained with PI. Fluorescent images were captured with a Leica DMRXA microscope at x 200 magnification. Controls omitting the first antibodies were all negative. D. Schematic illustration of induction time points points for microarray analysis and replicates at each time. E. Principal component analysis of induced RD-EF samples. F. Principal component analysis of tumor samples. G. Expression of EFT markers in RD-EF samples by microarray analysis. Red: T induced samples; Green: E induced controls. H. Expression of EFT markers in tumor samples by microarray analysis. 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RD-EF EF/Actin RD-EF Cyclin D1/Actin Time point;; (h) 6 12 18 24 Imlurer* j 2 2 4 I 2 2 s ' No. of RtfMntn E RD-EF 0 .0 » I IT " * 1 0 J 6 0 18 24 36 0 3 6 9 18 24 36 ■ T im e flit Tune (lit Nuclear Localization F Tumors T72h CD99 / CCK f STEAP/ - CD1 / / d ■ ' x" " / Ch«»*ev*t9W rari S T E W C y e fc D1 C-Myo I D 2 f c l ro r* « 2 EFT RMS NB 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. E-F expression in RD cells reveals a shift in global gene expression pattern with induction of EFT markers To provide visual representations of the samples based on gene expression, we used principal component analysis (PCA)(Golub, 1990) to locate the two-dimensional views that capture the greatest amount of variability in the data, using all 12,625 probe sets interrogating ^10,500 genes. The resulting PCA view showed that continued expression of the E-F chimeric gene induced a progressive shift away from both RD cells and EtOH induced controls (Figure 6E). All of the controls cluster together without an apparent pattern. However, Tet treated samples cluster in a distinct pattern that correlates directly with the induced time. Longer exposure to Tet resulted in a greater accumulation of E-F protein, and data points separated progressively further from that of the controls. These data indicated a fundamental difference in gene expression pattern between the E-F expressing cells and the controls. Detailed analysis of the differentially expressed genes revealed several noteworthy genes previously identified as EFT markers (CD99 (Ambros et al., 1991), CCK (Thiele, 1991), and STEAP (Hubert et al., 1999)), or E-F downstream genes (c-MYC (Dauphinot et al., 2001), ID2 (Fukuma et al., 2003; Nishimori et al., 2002) and CYCLIN D1 (Matsumoto et al., 2001)), all highly expressed in EFT (Fig 6H), and also highly up-regulated in the induced RD-EF cells (Fig 6G, Fig 4m-r). Interestingly, the up-regulation of c-MYC and ID2 (6h) preceded CYCLIN D1 (18h), which implied that these two genes are early response genes and CYCLIN D1 is possibly a secondary target of E-F. E-F down-regulates TGFpRII (Hahm et al., 1999) 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and P21 (Nakatani et al., 2003). It appeared that EtOH itself could regulate the expression of TGFPRII. However, Tet treated RD-EF cells showed more down- regulation than EtOH treated cells. P21 expression decreased dramatically up to 18h. Microarray expression patterns were found to be consistent with the real-time RT- PCR results, as demonstrated by the cyclin D1 result in Figure IB and 1G. Since the RNA for microarray and for real-time PCR analyses was from separate experiments, this consistency confirmed the reliability of the microarray results as well as the reproducibility of the RD-EF system. Thus, expression of E-F induces a marked shift in global gene expression, with induction and suppression of several known Ewing’s and EWS-FLIl target genes. Cultured RD-EF cells and RD-EF xenograft tumors that express E-F acquire the EFT phenotype. We monitored RD-EF cell morphology pre- and post- induction. Figure 7A illustrates the effect of only 24h’s expression of E-F in this cellular context. The spindle-shaped RD (typical for cell lines from skeletal muscle lineage) gradually became polygonal with frequent cellular processes. By 24h, the morphology of the induced RD-EF cells resembled EFT cells (TC 32) far more than untreated RD. Ethanol induction did not show this effect. This data was in accordance with the observation in C2C12-EF cells, (Eliazer et al., 2003) which exhibited a cuboidal appearance after E-F expression, and suggested that E-F expression mediated this morphological change of RD. 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The impression of probable neural induction observed by phase-contrast microscopy, was confirmed by electron microscopy and H&E staining of tumors derived from xenografts of RD cells, TC32 EFT cells, and RD cells expressing the E-F chimeric gene. RD cells normally form bona fide RMS tumors with marked terminal rhabdomyogenesis, while EFT cells are largely undifferentiated with scant neural differentiation marked by clusters of cytoplasmic dense core granules. Strikingly, RD cells that express E-F show near complete suppression of the myogenic phenotype with concomitant neural differentiation, marked by the appearance of neuritic processes containing dense core granules (figure 7B). H&E staining of RD-EF xenografts showed a structureless array of biphasic cell population (small dark and larger light types) with scant cytoplasm (Fig 9b), which is typical of EFT tumors (Fig 9c). On the other hand, RD xenografts still showed typical RMS tumor phenotype (a diffuse infiltrate of small round-to-spindled cells in a collagenous stroma, Fig 9a). Clearly, the E-F gene has a potent neural differentiating effect at the expense of normal tissue differentiation. This dual effect has not been previously recognized. 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 7. Cultured RD-EF with E-F expression and xenograft tumors formed by E-F expressing RD showed the phenotype of typical EFT. A. Light microscopy revealed a consistent morphological change after E-F induction in RD cells. B. EM study showed that xenograft tumors formed by E-F expressing RD cells lost muscle differentiation features that normally can be found in wild type RD formed tumors, and acquired certain degree of neural features, such as neurites and dense core granules. A. LM s .•I - j \ t s W g iM m 0 T12 B. EM RD (RMS) T18 TC-32 (EFT) T24 TC32 (EFT) RD + E-F eurite 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The appearance of an Ewing-like neuroectodermal phenotype is accompanied by diminished myogenic differentiation in induced RD-EF cells.A genetic basis for these observations was explored by gene expression analysis. Two cytoskeleton structural genes, microtubule associated protein tau (MAPT) and keratin 18, were up-regulated more than 8 fold after E-F expression (Fig 8Aa-b, p<0.001). MAPT is expressed exclusively in the axons of neurons and promotes microtubule assembly and stability. Both EFT and NB, but not RMS, expresses MAPT at high levels. Keratin 18 is an epithelial structural protein. The up-regulation of these two genes implied that E-F induced a neuroectodermal phenotype in mesodermal RD cells, consistent with the morphologic results reported above. Western blot analysis showed that the MAPT protein started to accumulate at 24h and reached peak level at 48h (Fig 8Ac, Fig 4j-l), which suggested that MAPT takes part in the morphological change induced by E-F expression. Moreover, the up-regulation of CCK (Fig 6G, Fig 9m-o, p<0.001), whose expression differentiates the parasympathetic neural phenotype of EFT from the sympathetic neural phenotype of NB, clearly indicated that a specific Ewing-like parasympathetic neural phenotype was imposed in RD cell by E-F expression. 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 8. The appearance of an Ewing-like neuroectodermal phenotype is accompanied by a depletion of myogenic differentiation in induced RD-EF A. Microarray (a), real time PCR (b) and western blot (c) analyses of MAPT after E- F induction in RD cells. B. Expression of important muscle differentiation associated genes in induced RD- EF cells (a) and in tumors samples (b) by microarray analyses and the down- regulation of MyoD and myogenin in induced RD-EF cells by western blot (c). B MAPT RD-EF MAPT/Actin £ 0 .0 6 S 0.04 3! 0.03 0.02 My 0 0 ^ 4 enip* : D esm in> CD3 Cliolhergic P.. 6 g.0.01 u j 0.00 0 3 6 9 18 24 36 M - — T »""" E Time (ID MAPT ACTIN r v j t j .r o P iV tyofltN h Cyolin D3 D esm in Cholinergic R « c « p to ra Cholinergic R ecep to r d M y f S M yf6 E-F MYOD MYOGENIN ACTIN RD-EF TC32 RD T24 T36 T48 T72 RD-EF We also examined the effect of E-F expression on myogenesis, cognizant of the profound loss of morphologic evidence of myogenesis noted in figure 7. Consistent with the morphology, we found a striking loss of expression of key genes necessary for myogenesis, especially the myogenic transcription factors MyoD and myogenin, the myogenic intermediate filament desmin, and muscle associated genes 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cholinergic receptor a and d. Two other myogenic transcription factors, myf5 and myf6, were unaffected by E-F expression (Fig 8Ba-b). Interestingly, this expression pattern of myogenic transcription factors is reminiscent of what was seen in biphenotypic sarcomas expressing E-F. Protein level confirmation of the down- regulation of MyoD and Myogenin by Western blot is illustrated in figure 3Bc, where a nearly inverse relationship between levels of E-F protein and these two transcription factors is noted. Moreover, Cyclin D3, the major D type cyclin expressed in RMS (Zhang, 2004) and associated with muscle differentiation (Bartkova et al., 1998; Cenciarelli et al., 1999), was down-regulated to an almost undetectable level after E-F induction, in parallel with the increase of cyclin D1 (the major D type cyclin in EFT). IHC staining is consistent with the microarray and Western results (Figure 9d-i). These data indicate that E-F expression induces a profound down-regulation of the muscle differentiation program. 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 9. IHC of xenografts. H&E staining (a-c) and immunohistochemistry (d-r) of Desmin, muscle specific actin (HHF35), MAPT, CCK and CD99 on xenograft tumors formed by RD, E-F expressing RD or TC32 cells. RMS RMS + EF EFT Comparison of up-regulated genes in RD-EF system and highly expressed genes in EFT revealed genes crucial in neural crest development. In an attempt to specify genes that were significantly up-regulated by E-F expression, we first identified a list of 865 “EFT signature genes” out of the 12,600 probe sets on the HU95av2 arrays, that are highly associated with primary EFTs from patients (p<=0.001) and are expressed at least 2 fold higher than in the rest of the tumors. PC A analysis showed that at time 24h and 36h, Tet induced RD-EF cells were completely separated from the controls and the EWS-FLIl protein level was stable and comparable with the original EFT cell lines. Thus we compared the gene 35 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. expression pattern of the T24 and T36 samples with all of the controls and selected 370 genes that are highly associated with the T24 and T36 samples (p<=0.001). Of these E-F induced genes, 109 (30%) are also signature genes of the EFT (Table 4, Fig 10B). Hierarchical clustering of the RD-EF samples separated these 109 genes in a temporal manner (Fig 10A). Two main gene expression patterns within the time course were identified: genes that are up-regulated at early time points (T6, T12), and genes that are up-regulated at late time points (T18, T24 and T36). We screened the gene lists by searching the literature as well as by their Gene Ontology Annotation. Strikingly, a marked number (30%) of E-F up-regulated genes are important for neural crest development, such as EGR2 (Krox20), Msxl, CITED2, c- myc, Id2, Cadherin 11, RUNX3, and Rho family members (ARHH (RhoH) and ARHGEF1). This is grossly disproportionate to the relative abundance of such genes by gene ontology code (p<0.001), and strongly suggests that a primary function of E- F is to invoke a form of neurogenesis. Other neural associated genes induced by E-F included neuronal pentraxin receptor, synaptotagmin I, SMA5, presenilin 1, as well as GABARAPL, XPNPEP1, DPYSL2, CSRP1, OLFM1, and many others (not listed here). Confirmatory RT-PCR was performed on selected genes (Figure 10C). The data showed a high correlation between microarray and RT-PCR results. In addition, we also used small interfering RNAs targeting the E-F breakpoint (siEFBP2, from Dohjima, et al (Dohjima et al., 2003)) to down-regulate E-F before induction. Twenty-four hours after transfection of siEF or a scrambled control C8, RD-EF cells 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. were induced by Tet or EtOH for another 24 hours before RNA was harvested. E-F expression level in siEF transfected cells induced by Tet was only 20% of those in C8 transfected cells induced by Tet. In accordance with the E-F level, the expression of identified genes was also lower in Tet induced siEF cells than the C8 controls (Fig 10D). This demonstrated that the expression of these identified genes, assessed by either microarray or QRT-PCR, showed the identical pattern and was E-F dependent. 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4.109 genes significantly upreguiated in RD-EF and also highly expressed in EFT. GenBank IE Affy ID Gene Name Symbol p Value T6 U65093 V00568 V00568 D13891 D13891 L19314 L20861 L20861 D31888 AL034562 L15388 X62822 AA978353 V01512 X76104 D21255 D21254 AL049801 AF070648 L29254 AB006537 M64174 M64174 X59372 X63657 AL039831 AF035752 AB011089 X83301 X75940 D82343 AC004770 W26480 AC004770 AF009767 AF009767 AF035284 M55047 AB020700 AF059274 AB020641 W28281 AB020643 AB018337 AL079294 ABO11004 AB018342 33113_at 37724_at 1973_s at 41215 s at Inhibitor of DNA binding 2, dominant negative helix-lo< ID2 41216_r_at 37393_at 31862_at 1669_at 37651_at 40144_at 1135_at 41352_at 41126 at Cbp/p300-interacting transactivator, with Glu/Asp-rich CITED2 V-myc myeiocytomatosis viral oncogene homolog (avian MYC Hairy and enhancer of split 1, (Drosophila) HES1 Wingless-type MMTV integration site family, member 5A WNT5 A 1916_s_at 40049_at 36976 at 2087_s_at 31790_at 36119_at 38763_at 38546_at 1457_at 41594_at 36882_at 36120_at 34877_at 339_at 39382_at 41643_at 41642_at 38855_s_at 41717_at 39372_at 41718 g at 41720_r_at 41719_i_at 39373_at 40075_at 35720_at 39966_at 36502_at 35785_at 33296_at 41691_at 39636_at 41242_at 35362 at REST corepressor RCOR Protein tyrosine phosphatase, non-receptor type substrate 1 PTPNS1 G protein-coupled receptor kinase 5 GPRK5 Sialyltransferase 1 (beta-galactoside alpha-2,6-sialyltransfe SI ATI Solute carrier family 1 (glutamate/neutral amino acid ti SLC1A4 T12 V-fos FBJ murine osteosarcoma viral oncogene homolog FOS Death-associated protein kinase 1 DAPK.1 Cadherin 11, type 2, OB-cadherin (osteoblast) CDH11 START domain containing 13 STARD13 Caveolin 1, caveolae protein, 22kDa CAV1 Sorbitol dehydrogenase SORD Interleukin 1 receptor accessory protein IL1RAP Janus kinase 1 (a protein tyrosine kinase) JAK1 Homeo box D9 HOXD9 Follicular lymphoma variant translocation 1 FVT1 Human HepG2 partial cDNA, clone hmd3f07m5. Caveolin 2 CAV2 Tripartite motif-containing 2 TRIM2 SMA5 SMA5 Olfactomedin 1 OLFM1 Fatty acid desaturase 1 FADS1 Synaptotagmin I SYT1 KIAA0893 protein KIAA0893 Chondroitin sulfate proteoglycan 5 (neuroglycan C) CSPG5 PFTAIRE protein kinase 1 PFTK1 GABA(A) receptor-associated protein like 1 GABARAF Likely homolog of mouse glucuronyl C5-epimerase GLCE KIAA0794 protein KIAA0794 Homo sapiens mRNA full length insert cDNA clone EUR( UDP-N-acteylglucosamine pyrophosphorylase 1 UAP1 Myosin X_________________________________________MYOIO 1.03E-12 3.28E-10 9.63E-10 2.80E-09 IE-08 8E-08 3.1E-07 7.4E-07 9.9E-07 0.000011 0.00008 0.0001 0.00036 7.33E-12 8.83E-10 IE-08 5E-08 IE-08 IE-08 2E-08 3.1E-07 6.4E-07 6.8E-07 7.9E-07 1.3E-06 1.4E-06 1.5E-06 5.6E-06 8.5E-06 0.00012 9.9E-06 0.000011 0.000013 0.000025 0.000075 0.0002 0.0004 0.000064 0.00012 0.00015 0.00017 0.00018 0.00023 0.00036 0.00039 0.00061 0.00064 38 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4.109 genes significantly upregulated in RD-EF and also highly expressed in EFT (Cont.) GenBank IE Affy ID Gene Name Symbol p Value T18 M69199 38326 at Putative lymphocyte G0/G1 switch gene G0S2 2.98E-13 AB014554 41435_at Protein tyrosine phosphatase, receptor type, f polypeptide (PPFIA3 1.54E-10 J03778 310 s at Microtubule-associated protein tau MAPT 2.27E-10 U94362 35334 at Glycogenin 2 GYG2 2.49E-10 M97676 40199 at Msh homeo box homolog 1 (Drosophila) MSX1 2.55E-10 M97676 215_g_at 2.3E-06 U28811 37706 at Golgi apparatus protein 1 GLG1 5E-08 M64788 1251 g at RAP1, GTPase activating protein 1 RAP1GA1 7E-08 M62403 1737 s at Insulin-like growth factor binding protein 4 IGFBP4 7E-08 U20982 3978l_at 3.60E-09 AF055581 39428 at Lymphocyte adaptor protein LNK 2.1E-07 AC005053 40297 at Six transmembrane epithelial antigen of the prostate STEAP 4E-07 X95762 35305 at X-prolyl aminopeptidase (aminopeptidase P) 1, soluble XPNPEP1 4.7E-07 Z35227 37416 at Ras homolog gene family, member H ARHH 8.6E-07 AJ243937 39049 at Chromosome 6 open reading frame 9 C6orf9 1.2E-06 X04297 32225 at ATPase, Na+/K+ transporting, alpha 1 polypeptide ATP1A1 1.8E-06 AF052182 39751 at Zinc finger, DHHC domain containing 3 ZDHHC3 2.5E-06 Y17448 36330 at Cysteine conjugate-beta lyase; cytoplasmic (glutamine tran CCBL1 2.9E-06 X14975 37861 at CD1E antigen, e polypeptide CD1E 0.000005 AF002672 38151 at Loss of heterozygosity, 11, chromosomal region 2, gene A LOH1 lCRi 8.6E-06 D83018 32598 at NEL-like 2 (chicken) NELL2 8.7E-06 X59798 38418_at Cyclin D1 (PRAD1: parathyroid adenomatosis 1) CCND1 0.000011 M73554 2020_at 0.000016 M64349 2017 s at 0.000053 X60673 3233 l_at Adenylate kinase 3 AK3 0.00026 L76528 642 s at Presenilin 1 (Alzheimer disease 3) PSEN1 0.00045 AB014530 39376 at Homeodomain interacting protein kinase 1-like protein Nbak2 0.00055 U66615 40810 at SWI/SNF related, matrix associated, actin dependent re SMARCC 0.00061 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4.109 genes significantly upregulated in RD-EF and also highly expressed in EFT (Cont.) GenBankIC Affy ID Gene Name Symbol p Value U05259 38017_at T24 CD79A antigen (immunoglobulin-associated alpha) CD79A 4.52E-13 U12255 31432_g_at Fc fragment of IgG, receptor, transporter, alpha FCGRT 4.14E-1I U12255 J04076 31431 at 37863 at Early growth response 2 (Krox-20 homolog, Drosophila) EGR2 4.40E-11 3.92E-10 X52560 38354 at CCAAT/enhancer binding protein (C/EBP), beta CEBPB 3.73E-09 W26700 36567_at 36567 at 2E-08 W26981 36568 at Homo sapiens cDNA FLJ33742 fis, clone BRAWH201901 4E-08 AB011126 40468 at Formin binding protein 1 FNBP1 2.3E-07 D25328 39175 at Phosphofructokinase, platelet PFKP 7.4E-07 U97105 40607 at Dihydropyrimidinase-like 2 DPYSL2 1.9E-06 M16279 41138_at CD99 antigen CD99 2.2E-06 Z35278 106 at Runt-related transcription factor 3 RUNX3 2.5E-06 M33146 38700_at Cysteine and glycine-rich protein 1 CSRP1 3.7E-06 AL008583 38836 at Neuronal pentraxin receptor NPTXR 5.6E-06 L37043 38019 at Casein kinase 1, epsilon CSNK1E 0.000007 AB018289 41585_at KIAA0746 protein KIAA0746 7.1E-06 U64105 810 at Rho guanine nucleotide exchange factor (GEF) 1 ARHGEF1 0.000021 AF067575 41222 at Signal transducer and activator of transcription 6, interleul; STAT6 0.000042 AB021663 39158 at Activating transcription factor 5 ATF5 0.000044 J04164 675 at Hypothetical protein MGC27165 MGC27165 0.000061 D42123 35828 at Cysteine-rich protein 2 CRIP2 0.00015 U07223 33244 at Chimerin (chimaerin) 2 CHN2 0.00023 ABO18267 32095_at Importin 13 IP013 0.00023 U43944 837 s at Malic enzyme 1, NADP(+)-dependent, cytosolic ME1 0.00033 AL049319 39172 at Hypothetical protein FLJ14547 FLJ14547 0.00074 D55649 41766_at Mannosidase, alpha, class 2A, member 2 T36 Lymphocyte antigen 96 MAN2A2 0.0011 ABO18549 33956 at LY96 0.000002 X07109 160029 at Protein kinase C, beta 1 PRKCB1 0.000047 L07765 37203 at Carboxylesterase 1 (monocyte/macrophage serine esterase CES1 0.00017 X56841 32321 at Major histocompatibility complex, class I, E HLA-E 0.00029 AW043690 37572 at Cholecystokinin CCK 0.00046 X98253 36565 at Zinc finger protein 183 (RING finger, C3HC4 type) ZNF183 0.0012 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 10. Comparison of up-regulated genes in RD-EF and highly expressed genes in EFT revealed important E-F target genes. A. Hierarchical clustering of microarray samples based on 109 genes common between EF induced genes in RD-EF cells and EFT signature genes. B-C. Selected genes that are highly expressed in EFT (B) and induced RD-EF cells (C-a) are validated by Real time PCR analysis (C-b). D. SiRNA targeting E-F breakpoint (EFBP2) or a control siRNA (C8) was introduced into RD-EF cells before tet or EtOH induction. Expression level of genes in C8 transfected, Tet induced cells was taken as 1. E. Van Diagram comparison of genes up-regulated in both RD-EF cells and HNFF- EF cells, as well as genes highly expressed in EFT cells revealed 46 genes common in all three groups. F. Identified E-F target genes that are also Known WNT signaling pathway targets. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T6 TI2 T18 T24T36 C B CeREsT EFT RMS m R M F IF 0 W it tU $hm O O O i I!|< H I H.N'FF- IT (|. stOlH> I5»nt#f4« (iM M ») two# that b v f k m iijih i*giiit«d in b»(h RD-IF and HNFF-1F. and a re also highly Nyrtmdln EFT, a re involved in gbdk 0 0 0 'f t 'ft M 111 ff> V Canonical Hon-Canonical CITED2 PTP R1A MSX1 WNT5A C-MYC PTPNS1 1 02 PKC |J1 CEBP|5 REST Co -Repressor Casein Kinase 1s Cyclin D1 Presenilinl 1.2 1.0 0 .# j- o.« - 0.4 ■ (- 0.2 0.0 Chip Data Q-RT-PCR CITED2 a TE D 2 s Act in 0.5 0.0 MSX1 2.5 2.0 1.5 1.0 0.5 0.0 CoRESTActin CoREST 0.9 0.4 NPR 2.5 2.0 1.5 1.0 0.5 0.0 JAK1 2.0 1.5 1.0 0.5 0.0 9 18 2436 RD-EF siEF EFA MSX1A CfTED2A CoRESTA MAPTA E3T24C8-24 0 T 2 4 E F E P 2 -2 4 D E24EFB P2-24 42 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Up-regulation of neural crest genes was confirmed in another cell lineage expressing E-F Our result suggests that E-F is a potent differentiation factor that blocks a pre existing commitment to myogenesis in RMS while imposing neural differentiation. We sought to determine whether neural differentiation is unique to the skeletal muscle cellular milieu found in RD cells, or is a generalizable effect of E-F. The fact that these genes are also highly expressed in primary EFT precludes the possibility that this is merely a nonspecific effect of the down-regulation of the muscle differentiation program. However, since cell context has been shown to be very important in extrapolating E-F’s function, we have pooled our RD-EF gene expression data with comparable expression profiles derived from primary, untreated EFTs and a similar dataset derived from E-F transfectants in a normal human foreskin fibroblast model kindly provided by the authors.(Lessnick et al., 2002) Using a cutoff of p < 0.001 (and expression > 2 fold increased in the case of primary EFT), we have performed a multiple t-test analysis of each dataset to identify the genes that meet these selection criteria, then compared them in a Venn diagram (Fig 10E) and identified a final 46 genes common to all three datasets (highlighted in table 4). These genes are of especial interest, because they are clearly up-regulated by expression of E-F, independently of cellular background, normal or malignant. Strikingly, the majority o f the neural crest differentiation related genes identified in RD-EF model were also up-regulated by E-F expression in this human fibroblast background. Fourteen of the 46 genes common to all three datasets are associated 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. with neurogenesis, and even more striking, of the 7 genes most strongly associated with the E-F expression in the model systems (e.g., p <0.000000001), the majority (5) are involved in neural differentiation, particularly the development of the neural crest (Table 4) (LaBonne and Bronner-Fraser, 1999). Thus, there is a strong association between neurogenesis and E-F expression. These data clearly indicate that neural differentiation induced by expression of E-F is independent of cellular background in which the gene is expressed. It excludes the possibility of a non specific neural up-regulation as a result of the down-regulation of myogenesis and indicates a general effect of E-F on differentiation. The canonical Wnt signaling pathway is not active in EFT To investigate the mechanism or signaling pathway(s) that E-F utilizes to affect the phenotype switch we observed (since it is an aberrant transcription factor), we inspected the 109 genes common to the RD-EF data set and the EFT primary tumor data sets. Strikingly, 20 of these up-regulated genes are well-known WNT signaling components, involving both canonical and non-canonical WNT signaling pathways (Figure 10F) (Nelson and Nusse, 2004). This observation suggested WNT signaling is an important mechanism whereby E-F invokes its biologic effect, especially since WNT signaling is important in the development of the neural crest, and aberrant WNT signaling has been implicated in diverse human cancers. We thus sought to confirm the likely role of WNT signaling in E-F medicated differentiation and potentially cell growth and metastasis. 44 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 11. Canonical Wnt signaling is not active in EFT and is not involved in the upregulation of EWS-FLI1 target genes A. Immunoflourescent microscopy of TC32 cells stained by a monoclonal antibody specific to human Fli-1 (red signal) and a polyclonal antibody specific to human B-catenin (green signal). Nuclei were counterstained with DAPI. Fluorescent images were captured with a Leica DMRXA microscope at x 200 magnification. Controls omitting the first antibodies were all negative. B. Luciferase reporter assay. C. Western blot analysis of B-catenin and phosphorylated B-catenin in induced RD- EF cells and tumor cells lines. DAPI ft-Catenin EWS-FLI1 X « *14 > 1 2 o 1 0 TC32 TC71 RD CHP126 ff{RD) V(RD) C S T O P □ FOP EWS-FLI1 p-catenln P- p-catenln Actln O 30 O x o ■ o o o A 5 w w n ■Si -. S T . 1 1 ST n RD-EF To investigate the canonical WNT/(3-catenin signaling pathway in EFT, we examined the cellular localization of (3-catenin in EFT cell lines by dual color fluorescence with DAPI (nuclear) counter-staining. Figure 11A reveals the result: whereas E-F protein was readily detected by immunoflourescent staining, localized Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. to the nucleus (red signal), P-catenin was found exclusively in the cytoplasm (green signal). This strongly suggested it was sequesetered in the cytoplasm. We also used a TCF-luciferase reporter assay (TOPflash/FOPflash) (Korinek et al., 1997) to detect activation of TCF-related gene expression by p-catenin binding to the reporter plasmid. Canonical WNT signaling is known to be active in NB as shown in figure 1 IB. However, there was no difference in luciferase activity between wild type (TOPflash) and mutated (FOPflash) reporter plasmids in EFT or RMS cell lines, indicating no P-catenin mediated gene expression, consistent with the cytoplasmic localization of P-catenin. As a final validation of these findings, phosphorylated vs. non-phosphorylated P-catenin was assessed on Western blots of NB (CHP126), RMS (RD), EFT (TC32 and TC71) cells, and compared to E-F expressing RD cells at 3 increasing time intervals (Figure 11C). E-F expression in RD cells down-regulated total p-catenin with increased phosphorylated p-catenin. Interpreted in light of all three experiments, there is no evidence that E-F utilizes a p-catenin-mediated, canonical WNT signaling pathway. 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Discussion The traditional view of oncogenes in tumor pathogenesis is to promote proliferation, increase survival and block the differentiation program of the target cells. However, chimeric fusion genes that result from various chromosomal translocations are different from traditional oncogenes in that they are often associated with specific tumor types, i.e. EWS-FLI1 in EFT, PAX3/7-FKHR in RMS and BCR-ABL in ALL. This specificity implies that the presence of the fusion gene may determine the differentiation program that the particular tumor type manifests. Transgenic mice models of Philadelphia chromosome positive (BCR-ABLpl90) acute lymphoblastic leukemia (Ph+-ALL) indicated that the target of leukemic transformation in Ph+-ALL is normal pluripotent hematopoietic stem cells rather than committed progenitor cells and the presence of BCR-ABLpl90 imposes the B cell differentiation program of the precursor stem cells but prevents further development of the committed B-cell precursors(Perez-Losada et al., 2001). It results in the accumulation of abnormal cells organized as a hierarchy, which failed to differentiate into functional B-lymphocytes. Similarly, when PAX3-FKHR was introduced into NIH3T3 cells, cDNA microarray analysis indicated profound activation of the myogenic transcription program, including upregulation of MyoD and myogenin (Khan et al., 2001). PAX3 transduced cells did not show this activation. Our study showed that EWS-FLI1 has a profound impact on cell differentiation as well as proliferation. It can induce a Ewing’ s-like neural crest 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. phenotype in multiple cellular contexts while blocking the existing cell differentiation program, which strongly supports the hypothesis that this tumor- specific fusion protein acts as a cell lineage determinator, rather than a pure “oncogene”. This represents an affirmation of a series of observations made by our group over the past several years. For the first time, however, we are able to extend the scope of our inquiry to assess the underlying mechanisms responsible for the observed phenotypic changes. Of particular interest is the profound effect on differentiation observed in RD RMS cells: otherwise conspicuous terminal skeletal muscle differentiation is nearly completely lost in RD cells expressing E-F, supplanted by marked neural differentiation as documented by the appearance of neurites, dense core granules, and a number of neural genes, as noted above. Krox20, MSXl(Streit and Stem, 1999), and c-MYC are all neural crest markers during development. Krox20 knock-out mice have severely defective myelination of peripheral nerves (Nagarajan et al., 2001). MSX1-/- mice have deficiencies in neural crest derivatives (Foerst-Potts and Sadler, 1997). Interestingly, forced expression of MSX1 in C2C12 cells reduced MyoD, myogenin, p21 and cyclin D3 protein to an undetectable level (Odelberg et al., 2000) and increased cyclin D1 expression level by 20-30 folds (Bafico et al., 2001). The similar pattern of regulation between E-F and MSX1 implied that the induction of these genes after E-F expression is at least partially mediated by MSX1. c-MYC and ID2 are previously identified E-F downstream response genes and it has been reported that ID2 is a direct target of c- MYC (Lasorella et al., 2000). Interestingly, c-MYC was shown to be an essential 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. early regulator of neural crest development and knockdown of c-MYC by antisense ODNs resulted in a loss of neural crest precursors in Xenopus embryos(Bellmeyer et al., 2003). Over-expression of ID2 can drive ectodermal cells into a neural crest phenotype instead of the epidermal lineage(Martinsen and Bronner-Fraser, 1998). CITED2 is a coactivator of another neural crest marker TFAP2. The expression of CITED2 is essential for the survival of neuroepithelial cells and disruption of CITED2 gene is embryonic lethal because of defects in the development of heart and neural tube(Ballard and Mikawa, 2002; Bamforth et al., 2001; Barbera et al., 2002). Cadherin 11 and Rho family GTPases are both related to delamination of neural crest cells from the neural tube(Adida et al., 1998; Borchers et al., 2001; Simonneau and Thiery, 1998). RUNX3 is an important regulator of the axonal projections of a subpopulation of dorsal root ganglion neurons(Inoue et al., 2002). Expression of neural structural genes, such as neuronal pentraxin receptor, symaptotagminl and MAPT, is also highly induced. CCK is a neuropeptide and was found to be highly expressed in EFT (Friedman et al., 1992). This is almost unique among tumor cells and served as evidence that EFT originated from parasympathetic progenitors. Another tumor type of neural crest sympathetic origin, neuroblastoma, does not express this gene. This was recently further corroborated by published observations that expression of E-F in a neuroblastoma cellular background (Rorie et al, 2004) suppresses sympathetic neural differentiation, the hallmark feature of neuroblastoma cells. Notably, MYCN, chromogranin A, and the TRK adapter protein ShcC were virtually eliminated, while MIC2 (CD99), a specific marker for 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. EFTs, c-MYC, and keratin 18 were all markedly up-regulated, similar to what we observed in our experiments. These data exclude the possibility of a non-specific neural up-regulation as a result of the down-regulation of myogenesis and indicate a general effect of E-F on differentiation. The high expression of all these critical neural crest associated genes provided further evidence of a parasympathetic neural crest differentiation program in EFT. More importantly, the fact that these genes could be highly induced by E-F implies that EWS-FLI1 is responsible for this observed neural crest phenotype of EFT. Since James Ewing described Ewing’s sarcoma more than 80 years ago, little has been known about the cell of origin of this group of tumors. Our results support the speculation that EFT probably originates from primitive multi-potent progenitor cells that are capable of differentiating into neural crest derivatives. EWS-FLI1 fusion subsequently imposes a neural crest parasympathetic lineage direction to the cells but inhibits terminal differentiation. Eventually secondary genetic alterations lead to the malignancy of the cells. Interestingly, EFT members represent a continuum of different degrees of neural differentiation. Bone marrow stromal stem cells (MSCs), which are classical mesodermal derivatives, have been shown to be multi-differentiated in addition to being multi-potent and could be induced to differentiate into neurons(Woodbury et al., 2002). Considering that most EFTs occur in bone and soft tissue, the MSC serves as one possible source of the cells susceptible of EWS-FLI1 transformation. A similar argument maybe true in soft tissue, where pluri-potent stem cells have also been described. 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Expression of EWS-FLI1 changed the transcription pattern of the RD cells, which is reminiscent of previously reported bi-phenotypic tumors with both neural and myogenic features.(Sorensen et al., 1995; Tan et al., 2001; Thomer et al., 1996) Only a myogenic differentiation program has been found to be able to co-exist with the neural features in cells that harbor the EWS-FLI1 (or EWS-ERG) translocation. Interestingly, PAX3 and PAX7, members of a paired-type homeobox (PAX) family of transcription factors and expressed transiently in myogenic and neural crest precursors during early stage of development, are both expressed and almost exclusively expressed in EFTs, RMSs, as well as biphenotypic sarcomas (Barr et al., 1999)(Tast, unpublished data). Loss of function analysis of PAX3 revealed that PAX3 is necessary for the proper formation of caudal neural crest derivatives and for the migration of myoblasts into the limb. Mice with a mutated Pax7 gene suffer from defects in cephalic neural crest derivatives.(Mansouri et al., 1996) The shared expression pattern of these two transcription factors in these tumors and the existence of bi-phenotypic sarcomas suggested close proximity between the programs of neural and skeletal muscle differentiation and a biological overlap of EFT and RMS. It is possible that these two groups of tumors originate from common PAX3 and PAX7 expressing cells and subsequent genetic alterations, such as EWS- FLI1 or PAX3/PAX7-FKHR, drives the cell to differing phenotypes. The suppression of muscle differentiation program and the induction of neural crest genes by EWS-FLI1 in RD cells provide evidence for this model. 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Despite several years of effort by multiple investigators, the basic mechanism whereby EWS-FLI1 expression results in the tumor entity that we recognize as Ewing’s sarcoma remains unknown. Little was revealed about how expression of a single chimeric “oncogene” can lead to such a profound genome-wide gene expression shift. This study does not permit us to distinguish direct from indirect targets of EWS-FLI1. However, by time course study and microarray analysis, we were able to infer a multitude of sequential downstream events and have identified an important but heretofore unidentified signaling pathway operative in Ewing’s tumors, both in vivo and in vitro (Fig 7). C-MYC and ID2 were among the earliest up-regulated genes and ID2 has been reported to be a direct target of both c-MYC and EWS-FLIl(Fukuma et al., 2003). It is possible that EWS-FLI1 can up-regulate both genes directly and over-expression of c-MYC can promote expression of ID2 to a higher degree. ID2 inactivates RB and subsequently promotes G1 phase transition. In addition, as a dominant negative bHLH transcription factor, ID2 antagonizes the expression of the myogenic transcription factors MYOD and MYOGENIN, as well as the cell cycle inhibitor P21. A previous study showed that P21 is a direct target of EWS-FLIl(Nakatani et al., 2003) and its expression was also down-regulated dramatically by EWS-FLI1 in our system. Another up-regulated gene, MSX1, an early neural crest marker during development, was found to be able to dedifferentiate terminally differentiated murine myotubes by repressing MyoD, Myogenin, cyclin D3, p21 and up-regulating cyclin D1 with minimal effect on cell proliferation (Bafico et al., 2001; Odelberg et al., 2000). Similar effects seen after EWS-FLI1 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. expression in RD cells lead us to speculate that EWS-FLI1 function is at least partially mediated by MSX1. However, MSX1 is not likely a direct target since its up-regulation starts at 18 hours after EWS-FLI1 expression, when MYOD, CYCLIN D3, and P21 were already down-regulated. A possible mediator of MSX1 up- regulation by EWS-FLI1 was CITED2, induced as early as 6 hours. It has been shown that MSX1 is a potent inhibitor of its own promoter region and this auto suppression could be counteracted by CBP/p300, which is the necessary partner of CITED2 for TFAP2 co-activation (Braganca et al., 2003; Mehra-Chaudhary et al., 2001). Figure 12. Gene regulation network initiated by EWS-FLI1. Genes in red were upregulated by EWS-FLI1. Genes in blue were downregulated by EWS-FLI1. Genes in square boxes are known Wnt signaling targets. N euro epithelium Survival Transform R ati Cells. An essential early regulator of nenal west cell formation in Xenopus. " S E W $ ^ F L I l < r . a l M i n f * t . ^ i . ■ i M m * i M i i m t ' " . . t a _ ' , t a iin g ' i — ------- CBP/p300, TFAP2 Forced M2 expression can convert ertodmnal cell to a neural crest fate Key factor dumig tlie development of nenral tube and netu ul crest V . / 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The striking incidence of WNT-signaling associated genes identified in this analysis can not be due to chance alone. WNT signaling has been found to be very important in neural crest development. (Garcia-Castro et al., 2002) WNTs could function as endogenous neural crest inducers in avian embryos, and inhibition of this pathway could block neural crest precursor formation in Xenopus (LaBonne and Bronner-Fraser, 1998). However, the classical WNT/p-catenin mediated signaling through TCF/LEF target genes is inactive in EFT tumors. Can EWS-FLI1 hijack the classical WNT signaling downstream of p-catenin? Or does it activate a non- canonical WNT pathway, whose mechanism is still largely unknown? Or has a completely different, currently un-described, pathway been utilized? Increased knowledge of the non-canonical WNT pathways will be required to answer these questions. 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Acknowledgements The authors would like to thank Darkin Chan and Minerva Mongeotti of the Electronic Microscopy Laboratory at Children’s Hospital-Los Angeles for EM processing; Morgan Wu of the clinical pathology laboratory at CHLA for IHC staining. We thank Betty Schaub, Xuan Chen and Sitara Waidyaratne of the Microarray Core Facility at CHLA for microarray analysis, and Dr. George Mcnamara at the CHLA imaging core for his expertise at microscopy. S.H. and part of this work was supported by an endowment in Molecular Pathology from Las Madrinas at CHLA. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Experimental procedures Transfection and Selection of Clones EWS-FLI1 type I fusion cDNA was cloned into pcDNA4/TO (Invitrogen,), a tetracycline inducible expression vector, and named pcDNA4/TO-EF. The construct was verified by DNA sequencing. Effectene (Qiagen) was used for all transfections according to manufacturer’s suggestions. To establish the tetracycline-regulated system (The TRex System, Invitrogen), RD cells were first stably transfected with pcDNA6/TR and monoclones were selected and maintained in tetracycline-free media containing 5 pg/ml Blasticidin (Invitrogen). The inducibility of each clone was tested by transient transfection with pcDNA4/TO/LacZ and then stained for P- gal using P-gal staining kit (Invitrogen). Two out of 40 pcDNA6/TR clones were chosen for second stable transfection with pcDNA4/TO-EF. Tetracycline-free media containing both 5 pg/ml Blasticidin and 400 pg/ml Zeocin (Invitrogen) was used for selecting and maintaining monoclones. Induction of EWS-FLI1 was accomplished by adding 1 pg/ml tetracycline to growth media and tested by RT-PCR and western blot. Tumors and Cell Lines Frozen tumor tissues were primary tumor tissues obtained prior to therapy from Childrens Hospital Los Angeles. All cell lines were obtained from our cell line bank and were cultured in RPMI with L-glutamine and 10% fetal bovine serum (Invitrogen/Gibco). Fusion gene status in primary tumor tissues and tumor derived cell lines were tested by RT-PCR. Among the 16 EFT, 36 RMS, 10 NB and 21 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. osteosarcoma primary tumor samples used for gene expression analysis, ET samples express either EWS-FLI1 or EWS-ERG, while the ARMS samples express either PAX3-FKHR or PAX7-FKHR. TC32 and TC71 are EFT lines with Type I EWS-FLI1 fusion. RD is an ERMS line. CHP126 is a NB line. Microarray Analysis For tumor samples, tissues that had more than 90% tumor cells were chosen. For cultured cell lines, RNA was harvested when cell confluence was 50-60%. Total RNA were extracted (RNA STAT-60, Tel-Test Inc.), cleaned (RNeasy mini kit, Qiagen), and quantitated. Synthesis of cDNA, biotin-labeled cRNA, fragmentation, target hybridization, washing, staining, and scanning probe arrays followed Affymetrix’s GeneChip expression analysis manual at CHLA microarray core facility. The commercially available Affymetrix HU95av2 arrays containing probes for approximately 12,600 human full-length genes and ESTs were used. Chip performance, background levels and presence/absence calls were first assessed using the Microarray Suite software (Affymetrix, Inc.). Cel files were normalized with ProbeProfiler software (Corimbia, Australia, www.corimbia.com) as described before (James et al., 2004). Bioinformatical analyses were performed with Genetrix analysis software (Epicenter Software). Real Time Quantitative RT-PCR Total cellular RNA was isolated using RNA Stat 60 (Tel-Test Inc) when cells reach 50-60% confluence. cDNA was synthesized from 2 pg of DNase I (Invitrogen) -treated total RNA in a 42 pi reaction volume using oligo-dT and Superscript II 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Invitrogen) for 60 minutes at 42°C following suppliers’ instructions. PCR primers were designed with MacVector 7.0 (Accelrys). The primer sequences are: Targets Forward Primers Reverse Primers MSX1 5 ’-TGCTCCAGTTTCACCTCTTTGC-3 ’ 5 ’-AACCTCTCTGCCCTCAGTTTCC-3 ’ EWS-FLI1 5 ’-CGACT AGTT ATGATCAGAGC AGT-3 ’ 5 ’-CCGTTGCTCTGTATTCTTACTGA-3 ’ CoREST 5'-ATGGCAACAGCAGCAGCAACTC-3’ 5’ -GGCAATGGCAATGTATTCATCC-3' NPR 5'-TGCTGGGTCAAGTGTCTCATCATAC-3' 5-CAAGTGCTGTC ACCTCCTTCCT AAG-3' JAK1 5'-CAGGTCTCCCACAAACACATCG-3' 5'-ACCAGGTCTTTATCCTCCAAGTAGC-3' CITED2 5'-TCTGTCTTGGCTTTGGCGTTC-3' 5'-ATTAGGGCGTTGAAGGCGTG-3' MAPT 5'-TGTGGCTCATTAGGCAACATCC-3' 5'-TCTGTCTTGGCTTTGGCGTTC-3' CYCLIN D1 5 ’-CGCACGATTTCATTGAACACTT-3 ’ 5 ’ -CGGATTGG AAAT AC TTCACAT-3’ p-ACTIN 5 ’ -GC ACCCCGTGCT GCTGAC-3’ 5 ’-C AGTGGT ACGGCC AGAGG-3 ’ PCR was performed on 1 pi of the cDNA in a final volume of 25 pi containing 0.5 pM of each primer and QuantiTect SYBR Green master mix (Qiagen). Real-time quantitative PCR was performed and analyzed using a SmartCycler (Cepheid) with SYBR green fluorescence signal detection after each cycle of amplification. Quantification was based on the cycle number necessary to produce a detectable amount of product above a defined threshold. Standard curves were constructed by four serial 10-fold dilutions of cDNA starting from 2 pi of the cDNA. PCR conditions were 95 °C 900 s; 40 cycles of 95 °C 15s, 55 °C (or 60C) 58 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 30s, 72 °C 30s; and a final denaturing stage by gradual increasing temperature from 60 °C to 95 °C. The denaturing stage is used to generate melting curves, which correlate with the size and GC content of the PCR products. Parallel internal control P-actin was amplified at the annealing temperature of 60 °C. To further ensure the specificity, all PCR products were analyzed on 1% agarose gel and single band of PCR product was observed in all reactions except negative controls. The reproducibility of the quantitative measurement of each sample was evaluated by at least three PCR measurements. The expression level of target gene was normalized to internal P-actin and the mean and standard deviation of the target/p-actin ratios were calculated for sample-to-sample comparison. Antibodies and Western Blot Analysis Monolayers of cells were lysed in immunoprecipitation buffer containing 50mM Tris HC1, 150mM NaCl, ImM EDTA, ImM EGTA, 25mM NaF, 25mM Beta-glycerolphosphate (PH 7.5), O.lmM sodium Vanadate, 5ug/ml leupeptin, 1 mM PMSF, 0.5% Triton X I00, and 0.5% NP40. Lysed cells were centrifuged for 15 min at 16,000g. Solubilized proteins in the supernatant were quantified using a BCA protein assay (Biorad), with BSA as a standard. Sixty pg of total cellular protein were loaded per lane, separated by pre-cast SDS-PAGE (Invitrogen), and then transferred to a PVDF membrane (Millipore, Bedford, MA). Blots were blocked with 0.5% I-Block in PBS, pH 8.0, with 0.1% Tween-20 before addition of primary antibodies and then horseradish peroxidase-conjugated secondary antibodies (Santa Cruz). Bound secondary antibodies were detected using an enhanced 59 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. chemiluminescence system (Amersham Pharmacia Biotech). The following polyclonal primary antibodies were used: MyoD, Myogenin and P-actin from Santa Cruz Biotechnology; p-catenin and phospho-p-catenin from Cell Signaling Technology. The following monoclonal primary antibodies were used: Fli 1 from BD Pharmingen and MAPT from Neomarkers. Transfections and Luciferase Assays Adherent TC32, TC71, RD and CHP126 cells were transfected by lipofectamine 2000 (Invitrogen) in six-well plates with a total of 4 pg of luciferase reporter constructs (Topflash or Fopflash system with a Renilla luciferase vector (SV40-pRL), ratio 50:1). The effects of EWS-FLI1 on the Topflash/Fopflash system were analyzed in RD cells. The cells were harvested and analyzed after 48 h. Luciferase assays were performed with the dual reporter luciferase assay kit (Promega). Immuno-fluorescent Staining To evaluate the expression and location of EWS-FLI1 and P-actin in TC32 and RD-EF cells, monolayer cells were grown in 4 well chamber slides and fixed in 2% paraformaldehyde at 4°C for 10 minutes followed by cold ethanol fixation at 4°C for 30 minutes. lOOmM glycine and 0.5% Triton-X 100 in PBS was used to permeabilize cells for 30 minutes at room temperature. After washing, sections were blocked with 5% normal donkey serum and 0.1% Tween 20 in PBS for 30 minutes and then incubated at 4°C overnight with Primary Abs against FLI1 (1:200, BD Pharmingen) and 13-catenin(1:400, Cell Signaling technology). Secondary antibodies, 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cy3-conjugated donkey anti-mouse or FITC- conjugated donkey anti-rabbit IgG (1:400, The Jackson ImmunoResearch, West Grove, PA, USA) were then applied and incubated in a dark chamber for 30 minutes. After washing with PBS, Mounting medium containing DAPI (Vector Labs) and a coverslip were applied (For nuclear localization of EWS-FLI1, FITC-conjugated donkey-anti-mouse IgG and mounting medium containing PI were used). Fluorescent images were captured with an Applied Spectral Imaging SD300/VDS1300 (Carlsbad, CA, USA) camera (using EasyFISH 1.2 software) mounted on Leica DMRXA (Bannockburn, IL, USA) microscope with Cy3 41004, GFP/Fluoresin 41001 and DAPI 31001 lenses (Chroma Technology Corp, Rockingham, VT, USA). Images were prepared for printing using Adobe Photoshop. Controls omitting the first antibodies were all negative. Xenograft Experiments. Four- to six-week old SCID mice were injected subcutaneously with 2 x 106 RD, TC32, or RD-EF leaky cells expressing comparable levels of EWS-FLI1 as EFT cell lines (n = 5). Tumor growth was monitored over time, and the mice were sacrificed when the tumors reach 1.5 cm in diameter. EF expression in the tumors was confirmed by real-time RT-PCR. Electron Microscopy Analysis Electron microscopic examination was carried out by the standard procedure at the electron microscopy laboratory, Childrens Hospital Los Angeles. Briefly, about 1 mm cubes from xenograft tumor samples were fixed with 2% glutaraldehyde in phosphate buffer (pH 7.4), post-fixed with 1% osmium tetroxide, and embedded in 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. epon (Embed-812, Electron Microscopy Sciences Inc.). One pm thick sections, cut from the hardened epon blocks and stained with 1% methylene blue and 0.5% basic fuchsin, were examined under the light microscope before ultastructural examination. Ultra-thin sections from the areas of interest were mounted on one- hole grids, stained with uranyl acetate/lead citrate, and examined and photographed with a Philip CM-12 transmission electron microscope. Immunohistochemistry Paraffin-embedded tissue blocks were sectioned (4-pm thick) onto slides and then deparaffinized. Sections were boiled 15 minutes in H2 0 for antigen retrieval, quenched with 3% hydrogen peroxide for 5 minutes. A Vector M.O.M. Immunodetection Kit (Vector Laboratories) was used for detecting monoclonal mouse-anti-human antibodies (MyoDl (DAKO), Myogenin (DAKO), Muscle Actin (Cell Marque), Desmin (DAKO), MIC2 (DAKO), and TAU (Neomarkers)) following the manufacturer’s protocol. A Vectastain Elite ABC Kit was used for polyclonal rabbit-anti-human antibodies (CCK (Neomarkers)). Slides were incubated in primary antibodies (1:100 dilution) 1 hour at room temperature, followed by incubation 30 minutes with biotinylated anti-mouse or anti-rabbit IgG secondary antibody. Sections were exposed to diaminobenzidine peroxidase substrate (Sigma) to give a brown stain and counterstained with Mayer’s hematoxylin. After washing with PBS and mounted, the sections were examined and photographed with a Nikon epifluorescent microscope (Japan). RNA Interference 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. siRNA targeting the breakpoint of E-F (siEFBP2) and Control siRNA (C8) were obtained from Dharmacon Research, Inc. (Lafayette, CO). Sequences of EFBP2 were from a previous report(Dohjima et al., 2003). SiEFBP2 or C8, complexed with TransIT-TKO (Mirus, Madison, WA) in Optimem I (Invitrogen Corp., Carlsbad, CA) according to the manufacturer’s directions, were applied to RD-EF cells 30% confluent in RPMI 1640 with 10% FBS but without antibiotics on six-well plates, to give a final concentration of 100 nmol/L. Twelve hours later, the medium was replaced with RPMI1640 with 10% FBS and RD-EF cells were induced by Tet or EtOH for another 24 hours before RNA was harvested. Gene expression was assessed by quantitative Real-Time PCR. 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 2 Secondary genetic alteration is required for EWS-FLI1 mediated oncogenesis. *Part of this work is published in Cancer Research. See appendix 1. Introduction The traditional view of the key global effect of the EWS-FLI1 chimeric transcription factor on the cellular phenotype has been to favor proliferation over differentiation in the putative neuroectodermal precursor cells of EFT, through transcriptional deregulation of target genes. As described in chapter one, we challenged this conception by showing that EWS-FLI1 has a lineage determination effect and can even impose a neuroectodermal phenotype to an otherwise already determined cell lineage. We want to further determine the effect of EWS-FLI1 on cell proliferation. Previous studies clearly showed that EWS-FLI1 is important in maintaining the tumorigenecity of EFT as abrogation of EWS-FLI1 in EFT tumor cell lines resulted in cell growth inhibition, loss of anchorage independent growth and absence of tumor formation in nude mice (Lambert et al., 2000; Ouchida et al., 1995; Tanaka et al., 1997). In addition, the EWS-FLI1 fusion type is correlated to the transactivation and proliferation rate of the tumors, i.g. type 1 EWS-FLI1 fusion, the most common type, is associated with a lower proliferative rate and a favorable prognosis (de Alava et al., 1998a). Furthermore, several recently identified direct or 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. indirect targets of the EWS-FLI1 fusion protein are involved in mitogenic signaling and cell cycle control, such as C-Myc, p57, PDGF-C, TGFBRII and Id2. NIH 3T3 cells can be transformed by forced EWS-FLI1 expression and can acquire the capability of anchorage-independent growth and tumor formation in immuno-deficient mice (May et al., 1993; Thompson et al., 1999). However, it was later discovered that simple transfection of the E-F gene was generally lethal, and certainly did not accelerate cell proliferation. Expression of EWS-FLI1 in normal mouse embryonic fibroblasts (MEF)(Deneen and Denny, 2001; Lessnick et al., 2002) resulted in apoptosis and cell growth arrest, preventing the establishment of stable expression of the protein in these cells. This EWS-FLI1 induced apoptosis and growth arrest were reduced in pl6/pl9ARF-null or p53 deficient MEFs. Lessnick (Deneen and Denny, 2001; Lessnick et al., 2002) showed that in telomerase-immortalized primary human fibroblasts, induced expression of EWS- FLI1 resulted in a non-senescent, non-apoptotic growth arrest, accompanied by upregulation of p53. Inhibition of p53, but not signaling upstream of p53, could rescue this growth arrest. This in fact has been true of most such oncogenes when simply transfected into normal or tumor cell backgrounds, for example, introduction of E l A or RAS induced an apoptotic or senescence response dependent on the p53/pl4ARF pathway. It indicates a cellular safety mechanism against the proliferative signals as a result of the EWS-FLI1 target gene deregulation and suggests that secondary genetic alteration is required to attenuate the growth arrest effect of EWS-FLI1. 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RD cells harbor P53 somatic mutation (Xia et al., 2002), therefore, we seek to investigate whether we can bypass the p53 induced cell growth arrest and promote cell proliferation by expressing EWS-FLI1 in RD cells. 66 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Results EWS-FLI1 increases cyclin D1 but decreases cyclin D3 expression in RD cells. D type cyclins play central roles in cell cycle Gl/S phase transition. In EFT, cyclin D1 is the major D type cyclin whereas in RMS Cyclin D3 is the major D type cyclin (Zhang, 2004). As mentioned in chapter one, after EWS-FLI1 induction in RD cells, cyclin D1 mRNA level increased dramatically while cyclin D3 mRNA level decreased to nearly undetectable level. On the other hand, there was no significant change of cyclin D2 expression associated with EWS-FLI1 expression. This result has been confirmed by Real Time RT-PCR analysis (Figure 13 A). To further analyze the roles of EWS-FLI1 on D-type cyclin expression and the effect of ERK1/2 activation in this process we followed the ERK1/2 phosphorylation status and the protein level of D cyclins at different time points after EWS-FLI1 induction. As shown in figure 13B and 13C, expression of EWS-FLI1 protein was first detectable 6 hours post induction and levels peaked by 36 hours. Significant increases in cyclin D1 protein were first observed at 18 hours post induction and continued to increase at subsequent time points. Although no changes in cyclin D2 were observed, expression of cyclin D3 start to decrease 6 hours after induction of EWS-FLI1 and was markedly reduced by 18 hours. In fact, cyclin D1 replaced cyclin D3 as the major D cyclin expressed in RD after induction of EWS-FLI1. ERK1/2 activities were only transiently up-regulated at early time points, likely due to changing of medium at 0 hour, and then decreased as EWS-FLI1 protein began to accumulate (Fig. 13C). Thus expression changes in cyclin D1 and D3 following EWS-FLI1 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. induction appeared to be independent of ERK1/2 activation. These data indicated that EWS-FLI1 specifically regulates cyclin D1 and D3 usage in tumor cells. Figure 13. EWS-FLI1 increases cyclin D1 but decreases cyclin D3 expression in RD cells. A. Real Time RT-PCR analysis of D type cyclins in induced RD-EF cells. B. Western blots detecting EWS-FLI1 protein, G1 cyclins and ERK1/2 phosphorylation at Thr202/Tyr204 (p-ERK) at different hours following treatment with either ethanol vehicle control (C) or 1 pg/ml tetracycline (T). Total ERK (t- ERK) was used as the loading control. C. Western blot band intensities in A were quantitated with FluorChem 8900. The intensity ratios of EWS-FLIl/t-ERK(EF/ERK), cyclin Dl/t-ERK(Dl/ERK), cyclin D3/t-ERK(D3/ERK), phospho-p44ERK/p44ERK(pERKl/ERKl), phospho- p42ERK/p42ERK(pERK2/ERK2) in ethanol control (C) or tetracycline (T) treated RD-EF are plotted. tm n o $ « # n u » - ♦ t f*mm 0 3 $ 9 18 24 M m ♦ m m tm rnl Tittle Rll m & tyctom m rn O h r 3hr S h i f t 3 9 9 WU M m ♦ tmm B 0 he fX f i IX fX x fX CT CT CT CT CT EWS-FLI1 C ycim D l C yclin P 2 Cyciui C y tim E l p~ERK o.t- 0.8 0 4 0.2 0 *SF*BK«T /%., *03fER«$-T 3 h r - t- fe h f Uhr - C t - 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. EWS-FLI1 expression did not change the CDK4 associated D cyclins in RD cells When cells enter the cell cycle from GO phase, D cyclins are the first induced cyclins and associate and activate cdk4 and cdk6 (Figure 14A). The cyclin/cdk complex subsequently target Rb for phosphorylation to promote G1 progression (Johnson and Walker, 1999). Therefore, we immunoprecipitated cyclin D-CDK4 complexes with anti-CDK4 antibody and then tested for the abundance of cdk4 associated D type cyclins after tet treatment of RD-EF cells using cdk4 as a loading control. As showen in figure 2B and 2C, after EWS-FLI1 expression in RD cells, although the expression of cyclin D1 and cyclin D3 is reversed, the level of the cdk4 protein and the level of cyclin D1 and D3 that associated with their catalytic partner cdk4 remained the same. These data indicated that the effect of EWS-FLI1 induction on cyclin D1 and D3 expression in RD cells is most likely changing the phenotype rather than promoting cell proliferation, i.e. EWS-FLI1 upregulated the major D type cyclin (Dl) in EFT and downregulated the major D type cyclin (D3) in RMS, with minimal effect on cdk4/cyclin D complex formation. 69 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 14. EWS-FLI1 expression did not change the CDK4 associated D cyclins in RD cells A. Regulation of E2F transcriptional activity through the cell cycle. (Adapted from Johnson & Walker, 1999) B. Western blots detecting D type cyclins and cdk4 protein levels of induced RD-EF cells. TC32 is a positive (EFT) control. C. Western blots detecting D type cyclins and cdk4 protein levels in cell lysates immunoprecipitated with anti-CDK4. A G O / <mrly G1 LateGI / early S S , G2, M E 2 F M S Repressor p21 1 cyclin E / cdk2 cyclin D / cdk4,S pie p53 p P P v S w Activator CD1 CD3 CDK4 Actin jc x: sz sz sz c m o O ) t co c\i n C N xf K Q RD-EF_ _ p21 cyclin A / cdk2 p Inactive C IP (CDK4) SZ SZ XZ JZ JZ 04 O 05 Xt 00 C M C O < M xf N Q — . r d -E F- H 70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. EWS-FLI1 expression in RD cells induced a non-apoptotic growth arrest. We also assessed whether EWS-FLI1 induction and its effects on cyclin D1 and D3 expression altered the proliferation of RD-EF cells by monitoring 3-day cell growth curve and comparing their cell cycle distribution with flow cytometry. RD-EF cells induced by EtOH showed the same proliferation rate as EtOH or tet induced RDVM cells (RD cells transfected by blank vectors without EWS-FLI1 cDNA sequence). On the other hand, the growth of tet treated RD-EF cells slowed down after 24 hours and the cell number was only half as much as the controls at 72 hours (Figure 15A). Flow cytometry analysis indicated that RD cells became arrested in G1 phase 24 hours after EWS-FLI1 induction (Figure 15B). However, there was no apoptotic peak observed in the FASC analysis and no significant cell death in the cell culture. Similar results were obtained with additional RD-EF clones (data not shown). Therefore, expression of EWS-FLI1 in RD cells induced a non-apoptotic growth arrest, similar to Lessnick’s observation when expressing EWS-FLI1 in immortalized human fibroblasts (Lessnick et al., 2002). Although EWS-FLI1 protein is required for ET cell proliferation as suggested by the anti-sense studies, it alone is not sufficient to promote cell proliferation. 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 15. EWS-FLI1 expression in RD cells induced a non-apoptotic G1 phase growth arrest. A. Cell growth curve after EWS-FLI1 induction of RD-EF cells. RDVM: Blank vector transfected control. B. FACS analysis comparing cell cycle distribution in un-treated (Blank), ethanol treated (ETOH-c), and tet treated (T-induced) RD-EF cells at 24 hour post-induction. R D V M E ■ -RDVM T m -- 1M i o Day 0 Day 1 Day! Day! B Blank %G0/G1*41.5 %G2M = 26 5 %S «32.0 ETOH-c %G0/G1*41 6 %G2M = 21 5 % $ *36.8 T-iatlnceti 14G0/G1* 75.3 % 02/M = 17 0 % S * 7 7 72 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. P21 protein level was up-regulated after EWS-FLI1 expression in RD-EF cells We next investigated the mechanism of EWS-FLI1 induced G1 phase growth arrest. As mentioned before, p53 is mutated in RD cells. Therefore, we detected the expression of members from two families of cyclin-dependent kinase inhibitors: pi 6 of the INK4 family, and p21, p27, p57 of the Cip/Kip family. P16 specifically interacts with cdk4 and cdk6 (Vogt PK, 1998), preventing the association of cdk4 and cdk6 with the D-type cyclins and inhibiting the activity of preassembled cyclin D/cdk4 and cyclin D/cdk6 complexes. As showed in Figure 16, EWS-FLI1 expression in RD cells resulted in a decrease of pi 6 protein level, therefore, pi 6 can not be the effector of EWS-FLI1 induced growth arrest. We next examined the protein level of the Cip/Kip family members, which can act on and inhibit most cyclin/cdk complexes at higher concentrations, and can function as assembly factors at lower concentrations for cyclin D/cdk4 complexes (LaBaer et al., 1997). P57 was reported to be an indirect target of EWS-FLI1 (Dauphinot et al., 2001) and re expression of p57 induced complete G1 arrest of Ewing’s sarcoma cells. p27 can inhibit transforming growth factor-p (TGF-P) and mediates contact inhibition (Lee et al., 1995). In RD-EF cells, both p57 and p27 level decreased after EWS-FLI1 expression. The p21 gene can be transcriptionally activated by p53 and thus a critical mediator of p53’s response to DNA damage. P21 inhibits cell cycle progression by inhibiting cyclin/cdk complexes and DNA synthesis by binding to the proliferating 73 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cell nuclear antigen (PCNA) subunit, an elongation factor for DNA polymerase 8(Li et al., 1994). In addition to p53, a variety of factors regulate gene transcription of p21, including Spl, Sp3, C/EBPs, and the STAT family. P21 has been reported to be a direct target of EWS-FLI1 (Nakatani et al., 2003). The protein level of p21 decreased at early time points after EWS-FLI1 induction in RD-EF cells (Fig 17), however, it’s expression went back and continue to increase at 24 hours, corresponding to the observation by cell growth curve and FACS analysis. Similar results have been documented in multiple independent experiments and different RD-EF clones. Since p53 is mutated in RD cells, this up-regulation of p21 is p53- independent. 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 16. P16 protein level decreased after EWS-FLI1 expression in RD. j T q — Inducible— o}' /C * c f * 0 T1d T2d T3d & & E-F p16 Id2 Actin Figure 17. Protein level of the Cip/Kip family of CKIs after EWS-FLI1 expression in RD-EF cells. I € ■ 1 i - 1 .... i In*** ifppi iHliP pp*“ v , j | + m m - * vppipppP 1 | V * < $ £ S ! > . S W S '- ' --^rnn ^ is ia s te . 1 | P K w S K B m ' , E T E T E T E T ^ Oh 1 8h 2 4h 3 6h 4 8h jr! EWS-FLI1 Cyclin D1 ] Cyclin D3 ] p27 ]p21 p57 Actin 75 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Discussion This study demonstrated that expression of EWS-FLI1 in embryonal rhabdomyosarcoma background upregulates the total cyclin D1 protein level and downregulates total cyclin D3 protein level. However, this D type cyclin expression shift did not affect the level of cdk4 associated cyclin D1 and D3 level, therefore is more likely an effect of EWS-FLI1 on phenotype determination rather than cell proliferation promotion. Though p53 is mutated in RD cells, EWS-FLI1 expression still induced a non-apoptotic cell growth arrest, similar to the result observed when EWS-FLI1 expression was induced in immortalized human fibroblasts. A possible explaination is the up-regulation of p21 in the induced RD- EF cells at advanced time points (24hours), though the RNA and protein level of p21 was down-regulated at early time points (less than 18hs). Future study by knocking-down p21 protein level before EWS-FLI1 induction in RD-EF will help to confirm the role of p21 in the EWS-FLI1 mediated growth arrest. These data support the hypothesis that the fusion protein by itself may be necessary but not sufficient for transformation. Previous study showed that nearly 60% of EFT demonstrated abnormal expression of at least one tumor suppressor protein, including pi 6 deletion (20-30%), p53 point mutation (10%), loss of p21 expression (55%), as well as pl4ARF(13%) and pi 5 (17%) abnormalities (de Alava et al., 2000; Lopez-Guerrero et al., 2001; Maitra et al., 2001; Patino-Garcia and Sierrasesumaga, 1997; Tsuchiya et al., 2000; Wei et al., 2000). p53/pl4ARF pathway mutation and pl6 deletion in EFT have a 76 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. markedly negative impact on disease specific survival and may also associate with advanced disease stage. In addition, reintroduction of wild type p53 in the p53-null EFT cell line SK-N-MC resulted in apoptosis in 70% of cells as compared to <10% of the empty vector control. Taken together, our data demonstrated that EWS-FLI1 functions more likely as a lineage determinator. It imposes an Ewing-like neural crest phenotype at the expense of the existing cell lineage and creates a permissive cellular environment for tumor development. However, EWS-FLI1 by itself is not sufficient and secondary genetic alterations are required for promoting cellular proliferation, therefore transforming the cells into EFT. 77 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Experimental procedures Real Time Quantitative RT-PCR Detailed procedures were described in chapter 1. The primer sequences for G1 cyclins are: D1F, 5 ’ -CGC ACG ATTTC ATT G A AC ACTT-3 ’; DIR, 5’-CGGATTGGAAATAC TTCACAT-3’; D2F, 5 ’ -TTGTCTCAAAGCTTGCCAGGA-3 ’; D2R, 5’-CGACTTGGATCCGTCAC GTT-3’; D3F, 5 ’-CCTCTGTGCTACAGATTATACCTTTGC-3 ’; D3R, 5’-TTGCAC TGCAGCCCCAAT-3’; PCR conditions were 95 °C 900 s; 40 cycles of 95 °C 15s, 55 °C 30s, 72 °C 30s; and a final denaturing stage by gradual increasing temperature from 60 °C to 95 °C. Antibodies and Western Blot Analysis Primary monoclonal antibodies against cyclin D l, D2, D3, E l, p57, p27, p21, pl6 and FLI1 c-terminal region were obtained from BD Bioscience. Polyclonal antibodies against CDK4, ID2 and P-actin were obtained from Santa Cruz Biotechnology. Phospho-ERKl/2 (Thr202/Tyr204), total ERK1/2 were obtained from Cell Signaling Technology. Immunoprecipitation Immunoprecipitation was performed as described before (Wu et al., 2001). Briefly, cellular protein lysates were first incubated with normal rabbit IgG and protein A+G. After spinning, the supernatant was quantitated and 800ug of protein lysates were 78 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. taken and incubated with rabbit anti-human CDK4. The cellular CDK4 complexes were then precipitated with protein A+G. After six times washing, the protein A+G conjugated CDK4 complexes were denatured in SDS-reducing buffer and then loaded on the protein gel for subsequent western blot analysis. Cell Proliferation Assays For cell growth curve analysis, the same number of cells was seeded in 12 well plates and cell number was counted every 24 hour for 3 consecutive days before reaching confluence. Each of EtOH-transfected or tet treated blank vector transfected or RD-EF had 3 wells for cell count. The distribution of cells in Go/Gi phase, S phase, and G2/M phase was determined by measuring DNA content as described before. Flow cytometric analysis was performed on FACStar (Becton Dickinson) and the cell cycle status was analyzed with Mac-cycle software. 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 3 Investigating the potential of targeting EWS-FLI1 fusion gene as an adjuvant therapy for EFT patients Introduction At diagnosis, approximately 30% of EFT patients already have detectable metastatic disease to lung (38%), bone (31%), eg. Spine, and bone marrow (11%). But in reality, nearly all patients have micrometastases, as with local therapy alone there is only a 10% cure rate. Metastasis to CNS, lymph nodes and liver is uncommon. The standard of care for EFT patients is systemic chemotherapy combined with surgery or radiotherapy for local control. Figure 18 shows the complexity of the conventional treatment of the EFT patients (Rodriguez-Galindo et al., 2003). Despite aggressive therapy, 40% of patients with localized disease and 80% of patients with detectable metastasis die due to tumor progression (Cotterill et al., 2000). Five-year survival rate is only approximately 50%. The most important negative prognostic factor of EFT is the presence of metastasis at the time of diagnosis (Terrier et al., 1996). Therefore, more specific and effective treatments are needed, especially for high risk (metastasized) cases.Our study and previous reports showed that EWS-FLI1 is the lineage determinator and initiator of EFT, and is important in maintaining the tumor growth. Abrogation of EWS-FLI1 by stably expressing anti-sense EWS-FLI1 in EFT cell lines resulted in decreased cell proliferation and loss of anchorage independent growth and tumorigenicity (Lambert et al., 2000; Ouchida et al., 1995; Tanaka et al., 1997). In addition, since EWS-FLI1 80 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. is only expressed in the EFT tumor cells, it serves as an ideal candidate for specifically targeting the minimal amount of disseminated tumor cells after the conventional treatment of the disease. Therefore, we want to establish an in vivo metastasis model of EFT and test if knockdown of EWS-FLI1 in vivo can be potential adjuvant therapeutics for EFT patients with metastasis. Figure 18. Conventional treatment of EFT (Adapted from Cotterill et al. 2000). Non-Pelvic and < 200 ml. and < 18 years and Type I W S -F U i Standard Risk Interm ediate Risk Pelvic or > 200 m l. or > 18 years or Lung metastases or Type II EWS-FIJ] High Risk Bone or bone marrow metastases Phase D Window Therapy Good Response: CHR Poor Response; PUR VCD VCLVIfc • Good Response: PR by imaging • Poor Response: < PR by imaging Good Response; GHR Poor Response: PHR Good Response; PR by imaging Poor Response: < PR by imaging VCD/IE WLI Uusulfan Mser + - ■ - w u VCU/jE w *t [ t u A u i r a n HSCT + < - W LI LOCAL C O NTR OL VCD/Its •♦-/-Phase il aecnt Busultan IfSCT < •/- WLI Other: C H . p o l ro P0CCT779 p o ro p o Abbreviations: VCD: vincristine, cyclophosphamide, doxorubicin; RT: radiation therapy: GHR: good histologic response; PHR: poor histologic response; PR: partial response; It: ifos&mide and etoposidc; WLI: whole lung irradiation; IISCT: hematopoietic stem cell transplant; CTL: cytotoxic T lymphocytes; ETO: etoposidc. TOPO: topotecan; PO: oral RNA interference (RNAi) is a post transcriptional gene silencing process mediated by double strand RNA to destroy the mRNA corresponding to this dsRNA sequence (Bass, 2001; Zamore, 2001; (Novina and Sharp, 2004)). It works efficiently in invertebrates such as C. elegans and drosophila, and is highly specific, very potent and really simple. The dsRNA can be delivered to C elegans by merely soaking or 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. feeding. The use of RNAi in somatic mammalian cells had been limited because of a nonspecific response of the cells to long dsRNAs (more than 30bp), which leads to a global inhibition of protein synthesis and degradation of all mRNAs (Figure 19A, left). However, this problem is solved by a recent discovery that RNAi can be mediated by 21-23 nucleotide dsRNA (siRNA) in cultured mammalian cells without inducing the sequence nonspecific effects (Figure 19A, right) (Caplen et al., 2002; Elbashir et al., 2001; Harborth et al., 2001; Paddison et al., 2002). The sense strand is degraded and the antisense strand is incorporated directly into RISC (RNA-induced silencing complex) to target a complementary mRNA for silencing (Figure 19B). Activated RISC (bound to the antisense strand) has sequence-specific mRNA- binding properties and participates in repeated cycles of degradation of specific mRNAs, therefore effectively silencing the gene from which the mRNAs are produced. It does not work as potently as in worms or flies but is still more efficient and stable than the traditional reverse genetic analysis, i.e. anti-sense or synthetic ribozyme technology. Our previous study shows that when introduced in vivo, naked siRNA does not induce immune response in mice (Heidel, et al, submitted, Appendix 2). Therefore, siRNA can be developed into a specific, potent but safe strategy to treat diseases. 82 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 19. RNA interference — post-transcriptional gene silencing. A. Mammalian cells have at least two pathways that compete for dsRNA. Adapted from Bass, 2001. B. Mechanism of siRNA mediated gene silencing. Adapted from Novina, 2004 Long double-stranded RNA >30 ta w palm Short interfering RNAa -19 base pairs m iiiti n Cleavage Nonspeelc effects • Degradation of all mRNA; : ► Inhibition of ell protein synthesis RNAI with sequence-) specie effects g « Degradation of sp e c ie mRNA B Vtorm sand plants Serree strand degradation /%igpSB0fflKA. FruiCiios end m a m m a s RISC ' Y ▼ target mRNA yvs®io(iAi/\ mRNA degradation Target mRNA Y f fflH N A mRNA degradation 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Systemic administration of siRNA demands a vehicle that can protect the siRNA and deliver it to the disseminated target sites. Thus, we have collaborated with Dr. Mark Davis’ group from the department of Chemical Engineering at California Institute of Technology, where they have developed a non-viral cyclodextrin (CDP)-based polymer delivery system with no immunogenicity and can be modified with ligands for cell targeting (Bellocq et al., 2003). In this system, cyclodextrin-containing polycation condenses siRNA into nanoparticles (Fig 20A) and poly-ethylene glycol-adamantane (PEG-AD) conjugates stabilizes the particles to minimize interactions with the plasma (Fig 20B). Transferrin (Tf), an iron-binding glycoprotein, was used for targeting the metastasized EFT. Tf is a well-defined ligand for tumor targeting and our preliminary data showed that TC71, the cell line we will use to establish the metastasis model in mice, highly express transferrin receptors (Tf-R). Figure 20. Structure of the key components of the delivery system. Adapted from Bellocq, 2003. A. (3-Cyclodextrin Polymers OH 'Ml B. Tf-PEG-Ad A sti- CARBOHYDRATE T ra n s fe rrin H •N \ N PEGjooo H 84 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. By using a combination of lipid transfection strategy and FACS sorting, Dohjima was able to show that endogenous expression of siRNA targeting the breakpoint of EWS-FLI1 fusion gene from a pol III promoter in a EFT cell line TC135 reduced the EWS-FLI1 transcript up to 80% and resulted in growth inhibition of the cells (Dohjima et al., 2003). In this study, we used the same siRNA sequences and tested if systemic administration of fully formulated siRNA targeting the breakpoint of EWS-FLI1 can cause metastasized EFT tumors to regress in vivo. 85 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Results In vitro study of the siRNA delivery to TC71 EFT cell line We first confirmed the efficacy of RNA interference in silencing gene expression in human cells by using a published siRNA sequence targeting lamin A/C (siLamin A/C). We transfected different concentrations of siLaminA/C to Hela cells using a lipid method (TransIT-TKO) and observed a dose-dependent knock-down of Lamin A/C protein up to 80% (Fig 21 A). lOOnM siLaminA/C could accomplish the highest efficacy without inducing toxic effects to the cells, as evidenced by no knock-down of the protein after transfecting lOOnM of unrelated siRNA control (FITC-labeled siRNA targeting luciferase, FITC-siLuc). Similar results were obtained by transfecting siLamin A/C to TC71 cells, an EFT cell line that expresses EWS-FLI1 fusion gene (Fig 21B). We next transfected lOOnM siRNA targeting EWS-FLI1 (siEFBPl and siEFBP2, sequence from (Dohjima et al., 2003)) to TC 71 cells and used a scrambled siRNA (siEFBP2Mut) and FITC-siLuc transfected cells as controls. For maximum efficacy, we transfected siRNAs on two consecutive days and harvested protein on the third day. As shown in Fig 21C, we did not observe a significant effect using siEFBPl, however, siEFBP2 transfection into TC 71 cells resulted in a strong reduction of EWS-FLI1 protein level, which was not observed with the control siRNAs. Therefore, siEFBP2 induced a sequence-specific down- regulation of the EWS-FLI1 protein level in TC71 cells. 86 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 21. In vitro study of the siRNA delivery to TC71 EFT cell line. A. Hela cells transfected with siRNA targeting Lamin A/C (siLamin). FITC-siLuc: FITC labeled siRNA targeting luciferase (negative siRNA control) B. TC71 cells transfected with siLamin. C. TC71 cells transfected with siRNA targeting EWS-FLI1 breakpoint (siEFBP). SiEFBP2Mut: scrambled siEFBP2, negative control Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A Hela siLam in A/C Lamin A/C a > * S s s £ a > c c c c C X O o o © o J * c T - c in c o V o r c ( 0 0 3 E ( 0 E m I ( 0 c E C E j -j jjj r a m o f ) ’ 3 5 t f ) d -j + + o f ) ' 3 5 3 3 + + 0 0 0 0 3 3 O O o o C M * * O H H * H o H Actin o o u > 6 H u. + 3 < N O * TC71 siLAMIN A/C * » $ % $ . |M r Lamin A/C t- O (- .* c « ffi > c o < z a < 5 5 > S 5 c c c O w o m 5 5 v* 35 — o < s ^ 3 < H “ 0 ) + C M T - o * tf) + 0 4 T ■ » o s c tf) + C M I 5 c 8 z tf) + C M T " s s c o o 5 c o o tf) + C M r ~ § < 0 + 0 0 O * I- Actin s £ e 5 5 o j 5 i • o h * iZ + C M T - o a: C TC71 siEFBP E-F Actin iEFBPI C M C L 0 0 LL LU 3 s C M C L C Q U 3 - J i o t r- C - O H j s c : c t f ) t f > L L L L (0 U l ' 3 5 C O Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Establishment of an EFT metastasis model With the help of Dr. Donald Kohn’s group at CHLA, we have established a TC71 EFT cell line (TC71-Luc) that stably express firefly luciferase using a lentiviral construct (Figure 22A). We injected these cells intravenously through tail vein to NOD/SCID immunodeficient mice, and monitored the tumor engraftment and growth using Xenogen in vivo imaging system. We first monitored the signal kinetics (peak and decay of the bioluminescent signal) after administration of luciferin (luciferase substrate). Two weeks after the injection of TC71-Luc cells, mice were given intraperitoneal injections of firefly luciferin at a dose of 150mg/kg. Images of the animals were acquired continuously beginning immediately after injection, each with a one-minute exposure. Peak signals were detected around 10 minutes after injection and stayed stable for another 10 minutes and then decreased rapidly over the next few minutes (data not shown). Therefore, all images in subsequent experiments were acquired 10 minutes after intraperitoneal injection of luciferin to standardize the measurement of signal in this model. The pattern of TC71-Luc cell engraftment was assessed by acquiring serial images over the course of 5 to 8 weeks after transplantation. Mice with no injection of TC71-Luc cells were used as negative controls. No detectable signal was apparent either before or after luciferin injection in the negative controls. However, signals in mice that received TC71-Luc cells could be detected by bioluminescence imaging right after the transplantation. Ten minutes after the injection, the signals accumulate in the lung area, indicating that most tumor cells were stuck in the capillary bed of 89 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the lung (Figure 22B). In the next few hours the luminescence signal starts to fade down and disappear, and start to appear again one to two week later at various locations. The most common engraftment sites are lung, vertebral column, pelvis, femur and soft tissue, which correspond to the popular metastasis sites of EFT tumors in patients. Figure 22. Establishment of an EFT metastasis model. A. The SMPU-R-MND-Luc Lentiviral Vector Provirus. B. NOD/SCID mice injected TC71-Luc cells developed metastasis tumors. A B 90 a U 3 AU3 10min ■ ■■ I II * * * R ite CTS 1. S = SIN LTR (*U 3) 2. M = Minimal gag/SD deleted 3. P * ePPT/CTS 4. U = UES polyA enhancer added 5. R =RRE added 6. MNO= MND LTR U3 promoter 7. Luc = Firefly Luciferase gene U3 R U5 ►AAA 3 days * « 10 days m* ttm « ¥ « ■ i ■ ■ x/m i Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Bioluminescent signal of the tumors decreased right after the fully formulated siLuc treatment. To test whether CDP-mediated siRNA transfer is valid for gene silencing in vivo, animal experiments were performed on mice bearing luciferase-producing metastasized EFT. Non-invasive in vivo bioluminescence imaging analysis showed that luciferase expressions in the tumor of mice injected with siLUC (GL3 siRNA) alone showed no inhibition. In contrast, mice administered with the siLuc/CDP complex showed a strong inhibition of luciferase expression in vivo at 2-3 days after injection, and increased thereafter (Figure 23 A and 23B). These results suggest that a CDP-mediated in vivo transfer of siRNA could be a powerful and simple method to mediate gene silencing in animals. Tumor growth was inhibited right after the fully formulated siEFBP2 treatment. Mice with successful engraftment of TC71-Luc cells were randomly selected for treatment with siEFBP2 siRNA alone or fully formulated siEFBP2/CDP-PEG-Tf. The tumor bioluminescence in mice is linearly correlated with the tumor volume(Rehemtulla et al., 2000; Vooijs et al., 2002). Metastasized EFT tumor growth was inhibited by treatment with siEFBP2 complexed with CDP-PEG-Tf. Consecutive injections of the formulated siEFBP for three days resulted in a delay of EFT tumor growth and this effect lasted 2-3 days (Figure 24A and 24B). In contrast, tumors treated with siEFBP2 alone and control siRNA/CDP-PEG-Tf showed no significant slow-down of the tumor growth. Therefore, the CDP-mediated siEFBP2 transfer is a significant novel method for inhibition of EFT tumor growth in vivo. 91 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 23. Bioluminescent signal of the tumors decreased right after the fully formulated siLuc treatment. A. Quantification of bioluminescent signals (photon flux) from a mouse that received formulated siLuc i.v. on day 41 and 42 after injection of TC71-luc cells, plotted against the number of days after transplantation. B. Images are of representative mice that received transplants of luciferase- expressing TC71 cells and were imaged serially before and after formulated siLUC treatment. All successive images in each row were taken from the same experimental mouse after transplantation at days shown. Exposure time was reduced for all animals at later time points as shown to avoid image saturation. Identical scales are used for image comparison. 92 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A TC71 -Luc Growth Curve siLuc Treatments o e t o 30 40 50 10 20 D ays after injection siLuc siLuc I I invi s — t.z>uf itjTiijj IR0I1 “4,7461 e+09i RQi 1-3.446te-ttl9 Color Bar Min = 1e+06 :Max= 1 e+OB Day 43 Day 44 Day 45 Day 47 93 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 24. Effect of siEFBP2/CDP complex on the growth of metastasized EFT. A. Quantification of bioluminescent signals (photon flux) from a mouse that received fully formulated siEFBP2 i.v. on day 29, 30 and 31 after injection of TC71-luc cells, plotted against the number of days after transplantation. ROI, Region of Interest. B. Images are of representative mice that received transplants of luciferase- expressing TC71 cells and were imaged serially before and after formulated siEFBP2 treatment. All successive images in each row were taken from the same experimental mouse after transplantation at days shown. Exposure time was reduced for all animals at later time points as shown to avoid image saturation. Identical scales are used for image comparison. 94 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A TC71-Luc Growth Curve 1 .2 0 1.00 0.80 siEFBP2 Treatments a o.eo 0.40 0.20 0.00 0 30 40 10 20 D ays after injection M03 Back ROI1 -B -M 0 3 Back ROI2 B siEFBP2 siEFBP2 siEFBP2 Color Bar Min- r.5e*»5 Day 32 Day 33 Day 36 Day 37 95 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Discussion Gene expression silencing by siRNAs is a powerful tool for the genetic analysis of a wide variety of mammalian cells and has the potential to be developed into a specific, potent and safe therapeutics for diseases. However, delivering siRNA into specific organs in vivo is a major obstacle for RNAi therapy. To overcome this problem, a hydrodynamic transfection method (high pressure tail vein injection) had been used in mice to deliver siRNA (and other type of nucleotides) to the liver. However, this method is not effective for other organs and is not feasible for clinical use (Song et al., 2003; Sorensen et al., 2003). Researchers have also shown successful use of viral-based plasmids for siRNA delivery to achieve prolonged and stable expression of siRNA (Hasuwa et al., 2002; Miyagishi and Taira, 2002; Pekarik et al., 2003); (Hemann et al., 2003); (Rubinson et al., 2003). Nonetheless, viral vectors suffer from the same severe side effects as gene therapy, which is still banned for clinical use. Therefore, the development of safe non-vector-based siRNA delivery systems is critical for the future of siRNA-based therapies. In this study, we first established a mouse metastasis model for Ewing family of tumors, which is highly reproducible with clinical relevance. The engraftment sites of the tumors correspond to the popular metastasis location in EFT patients. The use of an in vivo bioluminescent imaging system (Xenogen) allowed monitoring tumor growth over time in the same mouse. In addition, we showed that transferrin- conjugated cyclodextrin-mediated delivery of siRNA targeting EWS-FLI1 resulted in transient growth inhibition of metastasized EFT tumors. Because cyclodextrin 96 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. allowed increased cellular uptake, nuclease resistance and prolonged release of siRNAs, for clinical applications in RNAi therapy, a cyclodextrin-based siRNA transfer system represents an attractive method to achieve maximal function of siRNA-based gene silencing in vivo. Our delivery system also contained transferin ligands, which allowed site- specific transportation of siRNA to target sites. Thus higher concentration of complexes accumulates around the tumors to achieve maximal efficacy and reduce any adverse effects. Ligands targeting specifically to cell surface markers of EFT, for example MIC2, will be the focus of our future study. Although we did not see any immune reponse initiated by siRNA in our study (Appendix 2), recent reports has shown that siRNA sequences and their method of delivery may trigger an interferon response (Bridge et al., 2003; Sledz et al., 2003). Therefore, cyclodextrin-mediated non-vector transfer method is an attractive strategy to deliver siRNAs in vivo, since cyclodextrin has low toxicity and is low immunogenic, and hence unlikely to stimulate interferon expression in vivo. Finally, the in vivo gene silencing effect of siRNA by our delivery system is transient, evidenced by both siRNA targeting luciferase and EWS-FLI1. This transient feature permits fine-tuning of the intensity and interval of the treatment, i.e. the frequency of administration can be increased for stronger effect and the treatment can be stoped right away when needed. In the future, we will study administration of the formulated complex from earlier time points (for example, treat the mice twice a week right after the transplantation of the tumor cells), and combination of the 97 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. treatment with traditional chemotherapy, to investigate whether siRNA targeting EWS-FLI1 delivered by this cyclodextrin based system can have long term effect tumor growth and can be used as an adjuvant therapy for high risk patients of Ewing’s family of tumors. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Experimental procedures Antibodies and Western Blot Analysis Primary monoclonal antibodies against FLI1 c-terminal region were obtained from BD Bioscience. Polyclonal antibodies against Lamin A/C were obtained from BD Bioscience. P-actin were obtained from Santa Cruz Biotechnology. RNA Interference in vitro siRNA targeting lamin A/C, luciferase, the breakpoint of E-F (siEFBPl, siEFBP2) and Control siRNA were obtained from Dharmacon Research, Inc. (Lafayette, CO). Sequences of EFBP1 and EFBP2 were from a previous report(Dohjima et al., 2003). siRNA complexed with TransIT-TKO (Miras, Madison, WA) in Optimem I (Invitrogen Corp., Carlsbad, CA) according to the manufacturer’s directions, were applied to Hela cells or TC71 cells 30% confluent in RPM I1640 with 10% FBS but without antibiotics on six-well plates, to give a final concentration of 100 nmol/L. For siEFBPl and 2, siRNA-Trans-IT TKO complexes were applied again at 24 hours. All transfected cells were harvested at 48 hours and gene expression was assessed by Western Blot analysis. Lentiviral vector expressing the luciferase reporter gene SMPU-R-MNCU3-LUC is a lentiviral vector based on HIV-1 that transduces the firefly luciferase gene. The backbone vector SMPU-R has deletions of the enhancers and promoters of the HIV-1 long terminal repeat (LTR; SIN), has minimal HIV-1 gag sequences, contains the cPPT/CTS sequence from HIV-1, has 3 copies of the UES polyadenylation enhancement element from SV40, and a minimal HIV-1 RRE 99 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (gift from Paula Cannon, Childrens Hospital Los Angeles) (Bahner et al., 1996). The vector has the U3 region from the MND retroviral vector as an internal promoter (Challita et al., 1995) driving expression of the firefly luciferase gene from SP-LUC+ (Promega no. E178A; Promega, Madison, WI). Transduction of TC71 with luciferase vector TC71 cells were transduced with virus supernatant containing SMPU-R-MNCU3- LUC vector in the presence of interleukin 3 (IL-3), IL-6, and stem cell factor (SCF), as described (Case et al., 1999). A second cycle of transduction was performed 8 hours later by removing old medium and adding new virus supernatant and medium. Twenty-four hours after the initial transduction, cells were thoroughly washed 3 times with phosphate-buffered saline (PBS) before in vitro analysis. Injection of TC71-LUC (luciferase-expressing TC71) cells TC71-LUC Cells were grown in RPMI1640+10%FBS with PEN/STREP. Before injection, cells were trypsinized from the tissue culture flasks and washed twice with PBS. The viability of the cells was tested by adding 20ul trypan blue to 200ul of the cells and after a 5min interval counting 200-300 cells in a cell counter. Only cells more than 90% viable were used. For intravenous injection, 6 to 8-wk-old NOD/SCID mice were used. 5 x 106 TC71-LUC cells as single-cell suspension in 0.2 ml RPMI (without FBS or antibiotics) were injected through the tail vein of each mouse with a 27-gauge syringe. All experimental manipulations with the mice were performed under sterile conditions in a laminar flow hood. 100 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Delivery of siRNA to tumors in vivo Mice with successful tumor cell engraftment receive injection of siRNA-CDP complexes targeting luciferase, EWS-FLI1 or scrambled control on two or three consecutive days. SiRNA -CDP complexes were administered via the tail vein iv (50ug in 200ul of D5W in each case), using a 1-ml syringe and 27-gauge needle. Bioluminescent imaging of the mice After the injection, the mice were imaged at different time points using an in vivo prototype I VIS 3-dimensional bioluminescence/fluorescence optical imaging system (Xenogen, Alameda, CA). Luciferin (Xenogen) was injected intraperitoneally at a dose of 150mg/kg 10 minutes before beginning photon recording. General anesthesia was induced with 5% isoflurane in the light tight heated chamber. Anesthesia was continued during the procedure with 2% isoflurane introduced via nose cone. The imaging system consists of a cooled, back-thinned charge-coupled device (CCD) camera to capture both a visible light photograph of the animal taken with light-emitting diodes and the luminescent image, and a rotating mirror and translatable animal stage that allow for images to be acquired over 360° if desired. After acquiring photographic images of each mouse, anterior and posterior luminescent images were acquired with 1- to 3-minute exposure times. The resulting gray scale photographic and pseudocolor luminescent images were automatically superimposed by software so that identification of any optical signal with location on the mouse was facilitated. Optical images were displayed and analyzed with the IVIS Living Image (Xenogen) software packages. Regions of Interest (ROI) were 101 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. manually drawn around the bodies of the mice to assess signal intensity emitted. Optical signal was expressed as photon flux, in units of photons/s/cm2 /steradian. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 4 Concluding Remarks This is to our knowledge the first study to demonstrate a primitive neural crest phenotype for Ewing's Family Tumors consequent to expression of the tumor- specific EWS-FLI1 fusion gene. We compared global gene expression in Ewing's Family Tumors in vivo and in vitro with a time-course study of target genes identified by induced EWS-FLI1 expression using a T-Rex transfectant model in a rhabdomyosarcoma (RD) background. We paired this analysis with EWS-FLI1 targeted gene suppression using siRNA to independently verify these putative EWS- FLI1 target genes. We also demonstrated a similar phenotype in xenografts constitutively expressing the same EWS-FLI1 chimeric gene, and documented our results by histology, electron microscopy, and immunohistochemistry. As a result of the comprehensive scope of analysis, we were able to both validate known EWS- FLI1 target genes and identify new ones, and further to place them within the context of neural crest differentiation mediated by a novel form of WNT signaling. These data go farther towards elucidating the character of this enigmatic tumor than any previous study. These studies utilized patient tumor material as well as highly relevant in vitro models to validate these candidate genes, a good example of the emerging paradigm of bedside-to-bench research that allows real world validation of our laboratory studies. Likewise, because we used a standard gene expression platform 103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (e.g. Affymetrix GeneChips), we were able to reliably and informatively compare datasets from other investigators, which proved invaluable to these studies. From an onco-fetal developmental perspective, there has been a long standing debate along the lines of "seed or soil"- is Ewing's what it appears to be because of the (unknown) tissue of origin, or is it a consequence of an unknown function of the EWS-FLI1 gene beyond its role in oncogenesis (which is itself interesting in that EWS-FLI1 alone does not cause oncogenesis, as Lessnick's paper clearly documents). Thus, our study, for the first time, clearly and unequivocally demonstrates that it is "seed", not "soil"; the fact that expression of the chimeric TF in a committed rhabdomyogenic background extinguishes that phenotype, while inducing a neural crest phenotype, due solely and sequentially to expression of EWS- FLI1, has never before been shown. We thus finally explains what this enigmatic tumor is: a product of a single chimeric gene, operative in any permissive background. Given the 80+ years during which arguments have raged as to what this tumor is or arises from, our work will finally put this contentious issue to rest: Ewing's is what it is solely because of the phenotype induced by this chimeric transcription factor. It explains within a biologic context the true nature of this enigmatic tumor. Furthermore, we were able to cross confirm this by comparison with Lessnick's data, as well as primary tumor data compared to our in vitro system. This is also the first instance we are aware of where archival data has so nicely served to confirm new experimental data. Therefore, this will affect not only our 104 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. understanding of what is or is not a Ewing's tumor, but will also serve to define a new paradigm for tumor diagnosis and classification: it doesn't matter what it looks like, it only matters that an EWS chimeric gene must be operative within the tumor. This represents a true paradigm shift in cancer biology and diagnosis, with all that implies. Finally, we also established a highly reproducible mouse metastasis model for Ewing family of tumors. In vivo bioluminescent imaging system (Xenogen) allowed monitoring tumor growth over time in the same mouse. In addition, we showed that transferrin-conjugated cyclodextrin-mediated delivery of siRNA targeting EWS-FLI1 resulted in transient growth inhibition of metastasized EFT tumors in mice. Cyclodextrin-based siRNA transfer system represents an attractive method to achieve maximal function of siRNA-based gene silencing in vivo. Our study showed that fully formulated siRNA targeting EWS-FLI1 has the potential to be developed into an adjuvant therapeutic tool for treatment of EFT patients and prevention of tumor metastasis. 105 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Bibliography Adida, C., Crotty, P. L., McGrath, J., Berrebi, D., Diebold, J., and Altieri, D. C. (1998). Developmentally regulated expression of the novel cancer anti-apoptosis gene survivin in human and mouse differentiation. Am J Pathol 152, 43-49. Ambros, I. M., Ambros, P. F., Strehl, S., Kovar, H., Gadner, H., and Salzer- Kuntschik, M. (1991). MIC2 is a specific marker for Ewing's sarcoma and peripheral primitive neuroectodermal tumors. Evidence for a common histogenesis of Ewing's sarcoma and peripheral primitive neuroectodermal tumors from MIC2 expression and specific chromosome aberration. Cancer 6 7 ,1886-1893. Bafico, A., Liu, G., Yaniv, A., Gazit, A., and Aaronson, S. A. (2001). Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow. Nat Cell Biol 3, 683-686. Bahner, I., Kearns, K., Hao, Q. L., Smogorzewska, E. M., and Kohn, D. B. (1996). Transduction of human CD34+ hematopoietic progenitor cells by a retroviral vector expressing an RRE decoy inhibits human immunodeficiency virus type 1 replication in myelomonocytic cells produced in long-term culture. J Virol 70, 4352-4360. Bailly, R. A., Bosselut, R., Zucman, J., Cormier, F., Delattre, O., Roussel, M., Thomas, G., and Ghysdael, J. (1994). DNA-binding and transcriptional activation properties of the EWS-FLI-1 fusion protein resulting from the t(l 1 ;22) translocation in Ewing sarcoma. Molecular & Cellular Biology 14, 3230-3241. Ballard, V. L., and Mikawa, T. (2002). Constitutive expression of preproendothelin in the cardiac neural crest selectively promotes expansion of the adventitia of the great vessels in vivo. Dev Biol 251, 167-177. Bamforth, S. D., Braganca, J., Eloranta, J. J., Murdoch, J. N., Marques, F. I., Kranc, K. R., Farza, H., Henderson, D. J., Hurst, H. C., and Bhattacharya, S. (2001). Cardiac malformations, adrenal agenesis, neural crest defects and exencephaly in mice lacking Cited2, a new Tfap2 co-activator. Nat Genet 29,469-474. Barbera, J. P., Rodriguez, T. A., Greene, N. D., Weninger, W. J., Simeone, A., Copp, A. J., Beddington, R. S., and Dunwoodie, S. (2002). Folic acid prevents exencephaly in Cited2 deficient mice. Hum Mol Genet 11, 283-293. Barr, F. G., Fitzgerald, J. C., Ginsberg, J. P., Vanella, M. L., Davis, R. J., and Bennicelli, J. L. (1999). Predominant expression of alternative PAX3 and PAX7 forms in myogenic and neural tumor cell lines. Cancer Res 59, 5443-5448. 106 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Bartkova, J., Lukas, J., Strauss, M., and Bartek, J. (1998). Cyclin D3: requirement for Gl/S transition and high abundance in quiescent tissues suggest a dual role in proliferation and differentiation. Oncogene 17, 1027-1037. Bellmeyer, A., Krase, J., Lindgren, J., and LaBonne, C. (2003). The protooncogene c-myc is an essential regulator of neural crest formation in xenopus. Dev Cell 4, 827- 839. Bellocq, N. C., Pun, S. H., Jensen, G. S., and Davis, M. E. (2003). Transferrin- containing, cyclodextrin polymer-based particles for tumor-targeted gene delivery. Bioconjug Chem 14, 1122-1132. Bertolotti, A., Melot, T., Acker, J., Vigneron, M., Dellattre, O., and Tora, L. (1998). EWS, but not EWS-FLI-1, is associated with both TFIID and RNA polymerase II: interactions between two members of the TET family, EWS and hTAFII68, and subunits of TFIID and RNA polymerase II complexes. Molecular & Cellular Biology 18, 1489-1497. Borchers, A., David, R., and Wedlich, D. (2001). Xenopus cadherin-11 restrains cranial neural crest migration and influences neural crest specification. Development 128, 3049-3060. Braganca, J., Eloranta, J. J., Bamforth, S. D., Ibbitt, J. C., Hurst, H. C., and Bhattacharya, S. (2003). Physical and functional interactions among AP-2 transcription factors, p300/CREB-binding protein, and CITED2. J Biol Chem 278, 16021-16029. Bridge, A. J., Pebemard, S., Ducraux, A., Nicoulaz, A. L., and Iggo, R. (2003). Induction of an interferon response by RNAi vectors in mammalian cells. Nat Genet 34, 263-264. Brown, L. A., Rodaway, A. R., Schilling, T. F., Jowett, T., Ingham, P. W., Patient, R. K., and Sharrocks, A. D. (2000). Insights into early vasculogenesis revealed by expression of the ETS- domain transcription factor Fli-1 in wild-type and mutant zebrafish embryos. Mech Dev 90, 237-252. Burd, C. G., and Dreyfuss, G. (1994). Conserved structures and diversity of functions of RNA-binding proteins. Science 265, 615-621. Case, S. S., Price, M. A., Jordan, C. T., Yu, X. J., Wang, L., Bauer, G., Haas, D. L., Xu, D., Stripecke, R., Naldini, L., et al. (1999). Stable transduction of quiescent Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CD34(+)CD38(-) human hematopoietic cells by HIV-l-based lentiviral vectors. Proc Natl Acad Sci U S A 96, 2988-2993. Cavazzana, A. O., Miser, J. S., Jefferson, J., and Triche, T. J. (1987). Experimental evidence for a neural origin of Ewing's sarcoma of bone. Am J Pathol 127, 507-518. Cenciarelli, C., De Santa, F., Puri, P. L., Mattei, E., Ricci, L., Bucci, F., Felsani, A., and Caruso, M. (1999). Critical role played by cyclin D3 in the MyoD-mediated arrest of cell cycle during myoblast differentiation. Mol Cell Biol 19, 5203-5217. Challita, P. M., Skelton, D., el-Khoueiry, A., Yu, X. J., Weinberg, K., and Kohn, D. B. (1995). Multiple modifications in cis elements of the long terminal repeat of retroviral vectors lead to increased expression and decreased DNA methylation in embryonic carcinoma cells. J Virol 69, 748-755. Cotterill, S. J., Ahrens, S., Paulussen, M., Jurgens, H. F., Voute, P. A., Gadner, H., and Craft, A. W. (2000). Prognostic factors in Ewing's tumor of bone: analysis of 975 patients from the European Intergroup Cooperative Ewing's Sarcoma Study Group. J Clin Oncol 18, 3108-3114. Dauphinot, L., De Oliveira, C., Melot, T., Sevenet, N., Thomas, V., Weissman, B. E., and Delattre, O. (2001). Analysis of the expression of cell cycle regulators in Ewing cell lines: EWS-FLI-1 modulates p57KIP2and c-Myc expression. Oncogene 20, 3258-3265. de Alava, E., Antonescu, C. R., Panizo, A., Leung, D., Meyers, P. A., Huvos, A. G., Pardo-Mindan, F. J., Healey, J. H., and Ladanyi, M. (2000). Prognostic impact of P53 status in Ewing sarcoma. Cancer 89, 783-792. de Alava, E., Kawai, A., Healey, J. H., Fligman, I., Meyers, P. A., Huvos, A. G., Gerald, W. L., Jhanwar, S. C., Argani, P., Antonescu, C. R., etal. (1998a). EWS- FLI1 fusion transcript structure is an independent determinant of prognosis in Ewing's sarcoma. J Clin Oncol 16, 1248-1255. de Alava, E., Lozano, M. D., Sola, I., Panizo, A., Idoate, M. A., Martinez-Isla, C., Forteza, J., Sierrasesumaga, L., and Pardo-Mindan, F. J. (1998b). Molecular features in a biphenotypic small cell sarcoma with neuroectodermal and muscle differentiation. Hum Pathol 2 9 ,181-184. Dehner, L. P. (1993). Primitive neuroectodermal tumor and Ewing's sarcoma. Am J Surg Pathol 17, 1-13. Delattre, O., Zucman, J., Plougastel, B., Desmaze, C., Melot, T., Peter, M., Kovar, H., Joubert, I., de Jong, P., Rouleau, G., and et al. (1992). Gene fusion with an ETS 108 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DNA-binding domain caused by chromosome translocation in human tumours. Nature 359, 162-165. Deneen, B., and Denny, C. T. (2001). Loss o fp l6 pathways stabilizes EWS/FLI1 expression and complements EWS/FLI1 mediated transformation. Oncogene 20, 6731-6741. Dohjima, T., Sook Lee, N., Li, H., Ohno, T., and Rossi, J. J. (2003). Small interfering RNAs expressed from a Pol III promoter suppress the EWS/Fli-1 transcript in an Ewing sarcoma cell line. Mol Ther 7, 811-816. Eliazer, S., Spencer, J., Ye, D., Olson, E., and Ilaria, R. L., Jr. (2003). Alteration of mesodermal cell differentiation by EWS/FLI-1, the oncogene implicated in Ewing's sarcoma. Mol Cell Biol 23, 482-492. Ewing, J. (1921). Diffuse endothelioma of bone. Proc NY Pathol Soc 21, 17-24. Fellinger, E. J., Garin-Chesa, P., Triche, T. J., Huvos, A. G., and Rettig, W. J. (1991). Immunohistochemical analysis of Ewing's sarcoma cell surface antigen p30/32MIC2. American Journal of Pathology 139, 317-325. Foerst-Potts, L., and Sadler, T. W. (1997). Disruption of Msx-1 and Msx-2 reveals roles for these genes in craniofacial, eye, and axial development. Dev Dyn 209, 70- 84. Friedman, J. M., Vitale, M., Maimon, J., Israel, M. A., Horowitz, M. E., and Schneider, B. S. (1992). Expression of the cholecystokinin gene in pediatric tumors. Proc Natl Acad Sci U S A 89, 5819-5823. Fukuma, M., Okita, H., Hata, J., and Umezawa, A. (2003). Upregulation of Id2, an oncogenic helix-loop-helix protein, is mediated by the chimeric EWS/ets protein in Ewing sarcoma. Oncogene 22, 1-9. Garcia-Castro, M. I., Marcelle, C., and Bronner-Fraser, M. (2002). Ectodermal Wnt function as a neural crest inducer. Science 297, 848-851. Golub, G. H., van Loan, C. F. (1990). Matrix Computations (Baltimore, MD, Johns Hopkins University Press). Graves, B. J., and Petersen, J. M. (1998). Specificity within the ets family of transcription factors. Adv Cancer Res 75, 1-55. Grier, H. E. (1997). The Ewing family of tumors. Ewing's sarcoma and primitive neuroectodermal tumors. Pediatr Clin North Am 44, 991-1004. 109 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Hahm, K. B., Cho, K., Lee, C., Im, Y. H., Chang, J., Choi, S. G., Sorensen, P. H., Thiele, C. J., and Kim, S. J. (1999). Repression of the gene encoding the TGF-beta type II receptor is a major target of the EWS-FLI1 oncoprotein. Nat Genet 2 3 ,222- 227. Hartman, K. R., Triche, T. J., Kinsella, T. J., and Miser, J. S. (1991). Prognostic value of histopathology in Ewing's sarcoma. Long-term follow-up of distal extremity primary tumors. Cancer 6 7 ,163-171. Hasuwa, H., Kaseda, K., Einarsdottir, T., and Okabe, M. (2002). Small interfering RNA and gene silencing in transgenic mice and rats. FEBS Lett 532, 227-230. Hemann, M. T., Fridman, J. S., Zilfou, J. T., Hernando, E., Paddison, P. J., Cordon- Cardo, C., Hannon, G. J., and Lowe, S. W. (2003). An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo. Nat Genet 33, 396-400. Hubert, R. S., Vivanco, I., Chen, E., Rastegar, S., Leong, K., Mitchell, S. C., Madraswala, R., Zhou, Y., Kuo, J., Raitano, A. B., et a l (1999). STEAP: a prostate- specific cell-surface antigen highly expressed in human prostate tumors. Proc Natl Acad Sci U S A 96, 14523-14528. Inoue, K., Ozaki, S., Shiga, T., Ito, K., Masuda, T., Okado, N., Iseda, T., Kawaguchi, S., Ogawa, M., Bae, S. C., et al. (2002). Runx3 controls the axonal projection of proprioceptive dorsal root ganglion neurons. Nat Neurosci 5, 946-954. James, A. C., Veitch, J. G., Zareh, A. R., and Triche, T. (2004). Sensitivity and specificity of five abundance estimators for high-density oligonucleotide microarrays. Bioinformatics 20, 1060-1065. Johnson, D. G., and Walker, C. L. (1999). Cyclins and cell cycle checkpoints. Annu Rev Pharmacol Toxicol 39, 295-312. Khan, J., Wei, J. S., Ringner, M., Saal, L. H., Ladanyi, M., Westermann, F., Berthold, F., Schwab, M., Antonescu, C. R., Peterson, C., and Meltzer, P. S. (2001). Classification and diagnostic prediction of cancers using gene expression profiling and artificial neural networks. Nat Med 7, 673-679. Korinek, V., Barker, N., Morin, P. J., van Wichen, D., de Weger, R., Kinzler, K. W., Vogelstein, B., and Clevers, H. (1997). Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/- colon carcinoma. Science 275, 1784-1787. Kovar, H. (1998). Ewing's sarcoma and peripheral primitive neuroectodermal tumors after their genetic union. Current Opinion in Oncology 10, 334-342. 110 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LaBaer, J., Garrett, M. D., Stevenson, L. F., Slingerland, J. M., Sandhu, C., Chou, H. S., Fattaey, A., and Harlow, E. (1997). New functional activities for the p21 family of CDK inhibitors. Genes Dev 11, 847-862. LaBonne, C., and Bronner-Fraser, M. (1998). Induction and patterning of the neural crest, a stem cell-like precursor population. J Neurobiol 3 6 ,175-189. LaBonne, C., and Bronner-Fraser, M. (1999). Molecular mechanisms of neural crest formation. Annu Rev Cell Dev Biol 75, 81-112. Lambert, G., Bertrand, J. R., Fattal, E., Subra, F., Pinto-Alphandary, H., Malvy, C., Auclair, C., and Couvreur, P. (2000). EWS fli-1 antisense nanocapsules inhibits ewing sarcoma-related tumor in mice. Biochem Biophys Res Commun 279,401-406. Lasorella, A., Noseda, M., Beyna, M., Yokota, Y., and Iavarone, A. (2000). Id2 is a retinoblastoma protein target and mediates signalling by Myc oncoproteins. Nature 407, 592-598. Lee, M. H., Reynisdottir, I., and Massague, J. (1995). Cloning of p57KIP2, a cyclin- dependent kinase inhibitor with unique domain structure and tissue distribution. Genes Dev 9, 639-649. Lessnick, S. L., Braun, B. S., Denny, C. T., and May, W. A. (1995). Multiple domains mediate transformation by the Ewing's sarcoma EWS/FLI-1 fusion gene. Oncogene 10, 423-431. Lessnick, S. L., Dacwag, C. S., and Golub, T. R. (2002). The Ewing's sarcoma oncoprotein EWS/FLI induces a p5 3-dependent growth arrest in primary human fibroblasts. Cancer Cell 1, 393-401. Li, R., Waga, S., Hannon, G. J., Beach, D., and Stillman, B. (1994). Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair. Nature 371, 534-537. Lopez-Guerrero, J. A., Pellin, A., Noguera, R., Carda, C., and Llombart-Bosch, A. (2001). Molecular analysis of the 9p21 locus and p53 genes in Ewing family tumors. Lab Invest 81, 803-814. Mager, A. M., Grapin-Botton, A., Ladjali, K., Meyer, D., Wolff, C. M., Stiegler, P., Bonnin, M. A., and Remy, P. (1998). The avian fli gene is specifically expressed during embryogenesis in a subset of neural crest cells giving rise to mesenchyme. Int J Dev Biol 42, 561-572. I l l Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Maitra, A., Roberts, H., Weinberg, A. G., and Geradts, J. (2001). Aberrant expression of tumor suppressor proteins in the Ewing family of tumors. Arch Pathol Lab Med 125, 1207-1212. Mansouri, A., Stoykova, A., Torres, M., and Grass, P. (1996). Dysgenesis of cephalic neural crest derivatives in Pax7-/- mutant mice. Development 122, 831-838. Mao, X., Miesfeldt, S., Yang, H., Leiden, J. M., and Thompson, C. B. (1994). The FLI-1 and chimeric EWS-FLI-1 oncoproteins display similar DNA binding specificities. Journal of Biological Chemistry 269, 18216-18222. Martinsen, B. J., and Bronner-Fraser, M. (1998). Neural crest specification regulated by the helix-loop-helix repressor Id2. Science 281, 988-991. Matsumoto, Y., Tanaka, K., Nakatani, F., Matsunobu, T., Matsuda, S., and Iwamoto, Y. (2001). Downregulation and forced expression of EWS-Flil fusion gene results in changes in the expression of G(l)regulatory genes. Br J Cancer 84, 768-775. May, W. A., Lessnick, S. L., Braun, B. S., Klemsz, M., Lewis, B. C., Lunsford, L. B., Hromas, R., and Denny, C. T. (1993). The Ewing's sarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptional activator and is a more powerful transforming gene than FLI-1. Molecular & Cellular Biology 13, 7393-7398. Mehra-Chaudhary, R., Matsui, H., and Raghow, R. (2001). Msx3 protein recruits histone deacetylase to down-regulate the Msxl promoter. Biochem J 353, 13-22. Melet, F., Motro, B., Rossi, D. J., Zhang, L., and Bernstein, A. (1996). Generation of a novel Fli-1 protein by gene targeting leads to a defect in thymus development and a delay in Friend virus-induced erythroleukemia. Mol Cell Biol 16, 2708-2718. Miyagishi, M., and Taira, K. (2002). U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol 20, 497-500. Nagarajan, R., Svaren, J., Le, N., Araki, T., Watson, M., and Milbrandt, J. (2001). EGR2 mutations in inherited neuropathies dominant-negatively inhibit myelin gene expression. Neuron 30, 355-368. Nakatani, F., Tanaka, K., Sakimura, R., Matsumoto, Y., Matsunobu, T., Li, X., Hanada, M., Okada, T., and Iwamoto, Y. (2003). Identification of p21WAFl/CIPl as a direct target of EWS-Flil oncogenic fusion protein. J Biol Chem 278, 15105- 15115. 112 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Nelson, W. J., and Nusse, R. (2004). Convergence of Wnt, beta-catenin, and cadherin pathways. Science 3 03,1483-1487. Nishimori, H., Sasaki, Y., Yoshida, K., Irifune, H., Zembutsu, H., Tanaka, T., Aoyama, T., Hosaka, T., Kawaguchi, S., Wada, T., et al (2002). The Id2 gene is a novel target of transcriptional activation by EWS-ETS fusion proteins in Ewing family tumors. Oncogene 21, 8302-8309. Noguera, R., Triche, T. J., Navarro, S., Tsokos, M., and Llombart-Bosch, A. (1992). Dynamic model of differentiation in Ewing's sarcoma cells. Comparative analysis of morphologic, immunocytochemical, and oncogene expression parameters. Lab Invest 6 6 ,143-151. Novina, C. D., and Sharp, P. A. (2004). The RNAi revolution. Nature 430, 161-164. Odelberg, S. J., Kollhoff, A., and Keating, M. T. (2000). Dedifferentiation of mammalian myotubes induced by msxl. Cell 103, 1099-1109. Ohno, T., Ouchida, M., Lee, L., Gatalica, Z., Rao, V. N., and Reddy, E. S. (1994). The EWS gene, involved in Ewing family of tumors, malignant melanoma of soft parts and desmoplastic small round cell tumors, codes for an RNA binding protein with novel regulatory domains. Oncogene 9, 3087-3097. Ohno, T., Rao, V. N., and Reddy, E. S. (1993). EWS/Fli-1 chimeric protein is a transcriptional activator. Cancer Research 53, 5859-5863. O'Regan, S., Diebler, M. F., Meunier, F. M., and Vyas, S. (1995). A Ewing's sarcoma cell line showing some, but not all, of the traits of a cholinergic neuron. J Neurochem 64, 69-76. Ouchida, M., Ohno, T., Fujimura, Y., Rao, V. N., and Reddy, E. S. (1995). Loss of tumorigenicity of Ewing's sarcoma cells expressing antisense RNA to EWS-fusion transcripts. Oncogene 11, 1049-1054. Patino-Garcia, A., and Sierrasesumaga, L. (1997). Analysis of the pl6INK4 and TP53 tumor suppressor genes in bone sarcoma pediatric patients. Cancer Genet Cytogenet 98, 50-55. Pekarik, V., Bourikas, D., Miglino, N., Joset, P., Preiswerk, S., and Stoeckli, E. T. (2003). Screening for gene function in chicken embryo using RNAi and electroporation. Nat Biotechnol 21, 93-96. 113 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Perez-Losada, J., Gutierrez-Cianca, N., and Sanchez-Garcia, I. (2001). Philadelphia- positive B-cell acute lymphoblastic leukemia is initiated in an uncommitted progenitor cell. Leuk Lymphoma 42, 569-576. Rabbitts, T. H. (1994). Chromosomal translocations in human cancer. Nature 372, 143-149. Rao, V. N., Ohno, T., Prasad, D. D., Bhattacharya, G., and Reddy, E. S. (1993). Analysis of the DNA-binding and transcriptional activation functions of human Fli-1 protein. Oncogene 8 ,2167-2173. Rehemtulla, A., Stegman, L. D., Cardozo, S. J., Gupta, S., Hall, D. E., Contag, C. H., and Ross, B. D. (2000). Rapid and quantitative assessment of cancer treatment response using in vivo bioluminescence imaging. Neoplasia 2, 491-495. Rodriguez-Galindo, C., Spunt, S. L., and Pappo, A. S. (2003). Treatment of Ewing sarcoma family of tumors: current status and outlook for the future. Med Pediatr Oncol 40, 276-287. Rubinson, D. A., Dillon, C. P., Kwiatkowski, A. V., Sievers, C., Yang, L., Kopinja, J., Rooney, D. L., Ihrig, M. M., McManus, M. T., Gertler, F. B., et a l (2003). A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet 33, 401-406. Silvany, R. E., Eliazer, S., Wolff, N. C., and Ilaria, R. L., Jr. (2000). Interference with the constitutive activation of ERK1 and ERK2 impairs EWS/FLI-1-dependent transformation. Oncogene 19, 4523-4530. Simonneau, L., and Thiery, J. P. (1998). The mesenchymal cadherin-11 is expressed in restricted sites during the ontogeny of the rat brain in modes suggesting novel functions. Cell Adhes Commun 6, 431-450. Sledz, C. A., Holko, M., de Veer, M. J., Silverman, R. H., and Williams, B. R. (2003). Activation of the interferon system by short-interfering RNAs. Nat Cell Biol 5, 834-839. Song, E., Lee, S. K., Wang, J., Ince, N., Ouyang, N., Min, J., Chen, J., Shankar, P., and Lieberman, J. (2003). RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med 9, 347-351. Sorensen, D. R., Leirdal, M., and Sioud, M. (2003). Gene silencing by systemic delivery of synthetic siRNAs in adult mice. J Mol Biol 327, 761-766. 114 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sorensen, P. H., Shimada, H., Liu, X. F., Lim, J. F., Thomas, G., and Triche, T. J. (1995). Biphenotypic sarcomas with myogenic and neural differentiation express the Ewing's sarcoma EWS/FLI1 fusion gene. Cancer Res 55, 1385-1392. Sorensen, P. H., and Triche, T. J. (1996). Gene fusions encoding chimaeric transcription factors in solid tumours. Semin Cancer Biol 7, 3-14. Stout, A. (1918). A tumor of the ulnar nerve. Proc NY Pathol Soc 12, 2-12. Streit, A., and Stem, C. D. (1999). Establishment and maintenance of the border of the neural plate in the chick: involvement of FGF and BMP activity. Mech Dev 82, 51-66. Tan, S. Y., Burchill, S., Brownhill, S. C., Gerrard, M. P., Watmore, A., Wagner, B. E., and Variend, S. (2001). Small round cell tumor with biphenotypic differentiation and variant of t(21;22)(q22;ql2). Pediatr Dev Pathol 4, 391-396. Tanaka, K., Iwakuma, T., Harimaya, K., Sato, H., and Iwamoto, Y. (1997). EWS- Flil antisense oligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma and primitive neuroectodermal tumor cells. Journal of Clinical Investigation 99, 239- 247. Teitell, M. A., Thompson, A. D., Sorensen, P. H., Shimada, H., Triche, T. J., and Denny, C. T. (1999). EWS/ETS fusion genes induce epithelial and neuroectodermal differentiation in NIH 3T3 fibroblasts. Lab Invest 79, 1535-1543. Terrier, P., Llombart-Bosch, A., and Contesso, G. (1996). Small round blue cell tumors in bone: prognostic factors correlated to Ewing's sarcoma and neuroectodermal tumors. Semin Diagn Pathol 13, 250-257. Thiele, C. J. (1991). Biology of pediatric peripheral neuroectodermal tumors. Cancer Metastasis Rev 10, 311-319. Thompson, A. D., Teitell, M. A., Arvand, A., and Denny, C. T. (1999). Divergent Ewing's sarcoma EWS/ETS fusions confer a common tumorigenic phenotype on NIH3T3 cells. Oncogene 18, 5506-5513. Thomer, P., Squire, J., Chilton-MacNeil, S., Marrano, P., Bayani, J., Malkin, D., Greenberg, M., Lorenzana, A., and Zielenska, M. (1996). Is the EWS/FLI-1 fusion transcript specific for Ewing sarcoma and peripheral primitive neuroectodermal tumor? A report of four cases showing this transcript in a wider range of tumor types. American Journal of Pathology 148, 1125-1138. 115 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Torchia, E. C., Jaishankar, S., and Baker, S. J. (2003). Ewing tumor fusion proteins block the differentiation of pluripotent marrow stromal cells. Cancer Res 63, 3464- 3468. Truong, A. H., and Ben-David, Y. (2000). The role of Fli-1 in normal cell function and malignant transformation. Oncogene 19, 6482-6489. Tsuchiya, T., Sekine, K., Hinohara, S., Namiki, T., Nobori, T., and Kaneko, Y. (2000). Analysis of the pl6INK4, pl4ARF, pl5, TP53, and MDM2 genes and their prognostic implications in osteosarcoma and Ewing sarcoma. Cancer Genet Cytogenet 120, 91-98. van Valen, F., and Keck, E. (1988). Induction of glycogenolysis in cultured Ewing's sarcoma cells by dopamine and beta-adrenergic agonists. J Cancer Res Clin Oncol 114, 266-272. van Valen, F., Winkelmann, W., and Jurgens, H. (1992). Expression of functional Y1 receptors for neuropeptide Y in human Ewing's sarcoma cell lines. J Cancer Res Clin Oncol 118, 529-536. Vogt PK, R. S., eds. (1998). Cyclin Dependent Kinase (CDK) Inhibitors. (Berlin, Springer). Vooijs, M., Jonkers, J., Lyons, S., and Bems, A. (2002). Noninvasive imaging of spontaneous retinoblastoma pathway-dependent tumors in mice. Cancer Res 62, 1862-1867. Wei, G., Antonescu, C. R., de Alava, E., Leung, D., Huvos, A. G., Meyers, P. A., Healey, J. H., and Ladanyi, M. (2000). Prognostic impact of INK4A deletion in Ewing sarcoma. Cancer 89, 793-799. Weidner, N., and Tjoe, J. (1994). Immunohistochemical profile of monoclonal antibody 013: antibody that recognizes glycoprotein p30/32MIC2 and is useful in diagnosing Ewing's sarcoma and peripheral neuroepithelioma. Am J Surg Pathol 18, 486-494. Woodbury, D., Reynolds, K., and Black, I. B. (2002). Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis. J Neurosci Res 69, 908-917. Wu, L., Chen, P., Shum, C. H., Chen, C., Barsky, L. W., Weinberg, K. I., Jong, A., and Triche, T. J. (2001). MAT 1-modulated CAK activity regulates cell cycle G(l) exit. In Mol Cell Biol, pp. 260-270. 116 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Xia, S. J., Pressey, J. G., and Barr, F. G. (2002). Molecular pathogenesis of rhabdomyosarcoma. Cancer Biol Ther 1, 97-104. Zhang, J., Hu, S., Schofield, D., Sorensen, P., Triche, T.J. (2004). Selective Usage of D Type Cyclins by Ewing's Tumors and Rhabdomyosarcomas. Ca Res. Zucman, J., Delattre, O., Desmaze, C., Plougastel, B., Joubert, I., Melot, T., Peter, M., De Jong, P., Rouleau, G., Aurias, A., and et al. (1992). Cloning and characterization of the Ewing's sarcoma and peripheral neuroepithelioma t(l 1 ;22) translocation breakpoints. Genes, Chromosomes & Cancer 5, 271-277. Zucman, J., Melot, T., Desmaze, C., Ghysdael, J., Plougastel, B., Peter, M., Zucker, J. M., Triche, T. J., Sheer, D., Turc-Carel, C., and et al. (1993). Combinatorial generation of variable fusion proteins in the Ewing family of tumours. EMBO Journal 12,4481-4487. Zwemer, J. P., and May, W. A. (2001). PDGF-C is an EWS/FLI induced transforming growth factor in Ewing family tumors. Oncogene 20, 626-633. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Appendix 1 Selective Usage of D Type Cyclins by Ewing’s Tumors and Rhabdomyosarcomas (Cancer Research 2004 Sep 1; 64(17):6026-34) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Selective Usage of D Type Cyclins by Ewing’s Tumors and Rhabdomyosarcomas Jingsong Zhang1 , Siwen Hu1 , Deborah E. Schofield1 , Poul H.B. Sorensen1 ’ 2, and Timothy J. Triche1 department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, California 90033; Departments of Pathology and Pediatrics, Children’s and Women’s Health Center of British Columbia, University of British Columbia, BC, Canada V5Z 4H4. Running Title: D cyclins in Ewing’s tumor and rhabdomyosarcoma Keywords: Ewing’s Tumor, Rhabdomyosarcoma, Pediatric cancers, EWS- FLI1, Cyclin D, Sarcoma/soft-tissue malignancies, Gene regulation and transcriptional control, Gene expression profiling Reprint requests should be addressed to Timothy J. Triche Tel: (323) 669-4516, Fax: (323) 667-1123, E-mail: triche@usc.edu 119 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Abstract The genetic mechanisms that control proliferation of childhood musculoskeletal malignancies, notably Ewing’s tumor (ET) and rhabdomyosarcoma (RMS), remain largely unknown. Most human cancers appear to overexpress at least one of the G1 cyclins (cyclin D1-D3, cyclin E l, and cyclin E2) in order to bypass normal regulation of cell cycle G1 progression. We compared the gene expression profiles of 7 ET and 13 RMS primary tumor samples and found over-expression of cyclin D1 in all 7 ET samples. In contrast, RMS samples expressed higher levels of cyclin D2, cyclin D3, and cyclin El. This was confirmed by quantitive RT-PCR and Western blot. The relative roles of RAS-ERK1/2 and PI3K-AKT pathways in the regulation of D-type cyclin expression in these tumors were then assessed. Inhibition of either pathway reduced expression of cyclin D1-D3 in RMS lines while only PI3K inhibitors blocked cyclin D1-D3 expression in ET lines. Furthermore, PI3K-AKT appeared to regulate D-type cyclin transcription in RMS lines through FKHR and FKHRL1. Finally, the role of ET associated EWS-FLI1 fusion gene in regulating D cyclin expression was studied. Inhibition of EWS-FLI1 expression in the TC71 ET line decreased cyclin D1 but increased cyclin D3 levels. In contrast, induction of EWS-FLI1 expression in the RD RMS line increased cyclin D1 while decreasing cyclin D3 expression. Our results demonstrate distinct regulation of D type cyclins in ET and RMS, and indicate EWS-FLI1 can modulate their expression independent of cellular backgrounds. 120 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Introduction Ewing tumors (ET) and rhabdomyosarcomas (RMS) are malignant tumors of the musculoskeletal system in children, adolescents, and young adults that often share a similar small round cell morphology but have different histogenetic origins, genetic features, and clinical behaviors (1). ET arises in either bone or soft tissues, exhibits primitive neural features (2), and likely originates from a pluripotent neural stem cell. It is characterized by the presence of specific chromosomal translocations that fuse the 5’ portion of the 22ql2 EWS gene with the 3’ portions of different ETS family genes (3). Among the observed fusion genes, EWS-FLI1 is present in nearly 85% of ET (4), while EWS-ERG gene occurs in the majority of the remaining 15% (5). RMS arises in soft tissues and is thought to originate from primitive skeletal muscle precursor cells (6). The embryonal subtype (ERMS) constitutes approximately two thirds of all RMS and has frequent loss of heterozygosity at the 11 pi 5 locus. The alveolar subtype (ARMS) is characterized by PAX3-FKHR and PAX7-FKHR gene fusions, which are present in approximately 60% and 20% of ARMS, respectively (6, 7). Despite extensive genetic characterization of ET and RMS, relatively little is known about the mechanisms underlying their uncontrolled proliferation. Many human cancers harbor genetic alterations that target key regulators of cell cycle G1 progression. Analysis of the RB and the p53 pathways has demonstrated frequent p53 mutations, and loss of expression of various cyclin dependent kinase (CDK) inhibitors in ET and RMS cell lines, but these alterations 121 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. are not observed in the majority of primary ET and RMS tumors (8-13). Moreover, no mutations in RB have been reported in ET or RMS tumors (9, 14). Among positive regulators of G1 progression, high levels of cyclin D1 have been reported in ET cell lines (10, 15). Gene expression profiling of ET, RMS, neuroblastoma, and Burkitts lymphoma cell lines as well as primary tumors with cDNA microarray further demonstrated high levels of cyclin D1 expression in ET and neuroblastoma but not in RMS or Burkitt lymphoma (16). According to this data (Reference 16, Fig. 3b) expression of cyclin D1 in nearly half of the RMS samples is similar to that of Burkitt lymphoma, which is known to express cyclin D3 and or D2 but not cyclin D1 (17). Therefore while over-expression of cyclin D1 may be a mechanism of cell cycle progression in ET, the roles of cyclin D1 as well as two other D cyclins, cyclin D2 and D3, in RMS in this process are less clear. The D type cyclins function as key sensors for mitogenic growth factors and their expression levels appear to be rate limiting for cell cycle G1 progression (18). In accordance with their important roles, many human cancers over-express at least one of the D-type cyclins by either amplification of D cyclin genes (CCND1-3) or aberrant mitogenic signaling which lead to up-regulation of their expression (19, 20). On the other hand, analysis of D cyclin promoters has revealed marked differences in their regulatory elements, which suggests that transcription of these genes is independently regulated (21, 22). Indeed, cell- and tissue-specific patterns of D type cyclin expression were reported (23-25). ET and RMS, which have different cells of 122 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. origin, and different genetic abnormalities, might therefore utilize D type cyclins differently. In this study, we analyzed the expression of D cyclins in ET and RMS primary tumors as well as tumor derived cell lines. We observed that while cyclin D1 was the predominant D type cyclin expressed in ET, RMS instead expressed cyclin D2 and cyclin D3 at high levels. We further demonstrated that both the RAS- ERK1/2 and the PI3K-AKT pathways could regulate the expression of all three D cyclins in RMS cell lines, while the PI3K-AKT pathway appeared to be the major regulatory cascade for D cyclin expressions in ET cell lines. Moreover, inhibition of EWS-FLI1 expression in an ET line and inducing EWS-FLI1 expression in a RMS line reversed their cyclin D1 and D3 expression patterns, suggesting that this chimeric transcription factor mediates, at least in part, the observed over-expression of cyclin D1 and low expression of cyclin D3 in ET. MATERIALS AND METHODS Tumors and Cell Lines Frozen primary pre-treatment tumor tissues were obtained from Childrens Hospital Los Angeles with full IRB approval for research use of anonymized specimens.. Fusion gene status in primary tumor tissues and tumor derived cell lines were tested by RT-PCR. Among the tumors used for gene expression profiling, the 7 ET all expressed either EWS-FLI1 or EWS-ERG, the 8 ARMS expressed PAX3- FKHR or PAX7-FKHR, and 5 ERMS primary tumor showed typical histology. 123 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Additional primary tumor samples (3 ET and 3 RMS) were used for gene expression data verification. ET96 and ETOO express EWS-FLI1, while ET98 expresses EWS- jERG. Among the RMS samples, 2 are ARMs expressing either PAX3-FKHR (RA97) or PAX7-FKHR (RA96). RDES, 6647, TC135, TC71, TC32, TTC466, and A4573 are ET lines. All ET lines express EWS-FLI1 except TTC466, which expresses EWS- ERG. Among the EWS-FLI1 expressers, TCI35, TC71, and TC32 have type I fusion; RDES and 6647 have type II fusion; and A4573 has type III fusion. RD, TC442, and Rhl8 are ERMS lines, while Rh28, Rh30, and TC487 are ARMS lines expressing PAX3-FKHR fusion gene. All the above cell lines were obtained from our cell line bank except RDES, which was purchased from the American Type Culture Collection (ATCC). All cell lines were cultured in RPMI 1640 containing 10% FBS (Invitrogen/Gibco). Expression Analysis with the U95Av2 Microarray Tissues that had more than 90% tumor cells were chosen and total RNA was extracted (RNA STAT-60, Tel-Test Inc.), cleaned (RNeasy mini kit, Qiagen), and quantitated. Synthesis of cDNA, biotin-labeled cRNA, target hybridization, washing, staining, and scanning probe arrays followed Affymetrix’s GeneChip expression analysis manual. To test the chip-to-chip variation, the same biotin-labeled cRNA from one ET sample was hybridized to three chips. Gene expression data was first analyzed with Microarray Suite software 5.0 (Affymetrix) and the average difference of each gene/probe set was then used to generate the text file for analysis with GeneSpring 5.0 (Silicon Genetics). When the 124 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. raw data was imported into GeneSpring, two normalizations were carried out. For each chip, the signal strength of each gene was normalized to the median of all the measurements taken in that chip. For each gene, the signal strength was normalized to the median of every measurement taken for that gene throughout all the chips imported into that experiment. All the negative values were forced to zero. In addition to generating the normalized expression level for each gene on each chip, the “trust” of each gene is calculated by multiplying the median value of the chip with the median value of the gene and interpreted by specifying 500 as most trust worthy, 150 as moderate trustworthy, and 50 as least trust worthy. Real Time Quantitative RT-PCR Total cellular RNA was extracted with RNA Stat 60 (Tel-Test Inc) when cells reach 70-80% confluence. For cDNA synthesis, 1 pg total RNA was first digested with amplification grade DNase I (Invitrogen) and then reverse transcribed with Superscript II (Invitrogen) following supplier’s instructions. cDNA sequences specific to each cyclin were selected and PCR primers were designed with Primer Express 2.0 (Applied Biosystems). The primer sequences for G1 cyclins are: DIF, 5’-CGCACGATTTCATTGAACACTT-3 DIR, 5 ’ -CGG ATT GGAA AT AC TTCACAT-3’; D2F, 5’-TTGTCTCAAAGCTTGCCAGGA-3D2R, 5’- CGACTTGGATCCGTCAC GTT-3’; D3F, 5’- CCT CTGTGCTAC AG ATT AT ACCTTT GC-3 ’; D3R, 5’-TTGCAC TGCAGCCCCAAT-3’; E1F, 5’-TGAAGAAATGGCCAAAATCGA-3’; E1R, 5’- AACC CGGTC ATC ATCTTCTTTGT-3 ’; E2F, 5 ’ - AGCCC AGCC AG ACGG A AT - 125 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3’; E2R, 5 ’ -C AG ATA AT AC AGGTGGCC AAC A AT-3 ’. The primer sequences for p-actin are 5’-GCACCCCGTGCTGCTGAC-3’ (forward) and 5’- CAGTGGTACGGCCAGA GG-3’ (reverse). For each PCR reaction, 1/20 (1 pi) of the cDNA reaction was amplified in a 25 pi reaction volume containing 0.5 pM of each primer and QuantiTect SYBR Green master mix (Qiagen). The real time quantitative PCR was carried out on SmartCycler (Cepheid). Standard curves were constructed by four serial 10-fold dilutions of cDNA starting from 1/10 (2 pi) of the cDNA reaction. PCR conditions were 95 °C 900 s; 40 cycles of 95 °C 15s, 55 °C 30s, 72 °C 30s; and a final denaturing stage by gradual increasing temperature from 60 °C to 95 °C. The denaturing stage is used to generate the melting curve of PCR products, which correlate with the size and GC content of the PCR product. Parallel internal control p-actin was amplified at the annealing temperature of 60 °C instead of 55 °C. All PCR products were ran on the 1.5% agarose gel and single band of PCR product was observed in all reactions except negative controls. The reproducibility of the quantitative measurement of each sample was evaluated by at least three PCR measurements. The mean and standard deviation of the cyclin/p- actin ratios were calculated for sample-to-sample comparison. Antibodies and Western Blot Analysis Protein lysate preparation and immunoblotting were performed as described before (26). Primary monoclonal antibodies against cyclin D l, D2, D3, El, and FLI1 c-terminal region were obtained from BD Bioscience. Monoclonal antibody against P-actin was obtained from Sigma. Polyclonal antibodies against cyclin E2, CDK2, 126 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CDK4, and FKHRL1 were obtained from Santa Cruz Biotechnology. Phospho-AKT (Ser473), total AKT, phospho-ERKl/2 (Thr202/Tyr204), total ERK1/2, phospho- FKHR (Ser256), phospho-FKHR (Thr24)/FKHRL1 (Thr32), total FKHR, and phospho-AFX (Seri 93) were obtained from Cell Signaling Technology. Secondary horseradish peroxidase conjugated goat anti-mouse IgG, goat anti-rabbit IgG, and donkey anti-goat IgG were obtained from Santa Cruz Biotechnology. The blotted proteins were visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech). Protein band intensities were quantitated with FluorChem 8900 (Alphalnnotech). Immunoprecipitation Immunoprecipitation was performed as described before (26). Briefly, cellular protein lysates were first incubated with normal rabbit IgG and protein A+G. After spinning, the supernatant was quantitated and lmg of protein lysates were taken and incubated with rabbit anti-human CDK4. The cellular CDK4 complexes were then precipitated with protein A+G. After six times washing, the protein A+G conjugated CDK4 complexes were denatured in SDS-reducing buffer and then loaded on the protein gel for subsequent western blot analysis. Kinase Inhibitor Studies ET and RMS cells were seeded and starved in RPMI 1640 medium containing 0.5% FBS. After 48 hours, medium was changed with 0.5% FBS medium with either the MEK1/2 inhibitor U0126 at 20 pM (Cell Signaling Technology) or the PI3K inhibitor LY294002 (Cell Signaling Technology) at 50 pM or equivalent 127 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. volume dimethylsulfoxide (DMSO) vehicle control. At different time points following treatment, total RNA and protein lysates were harvested for real time RT- PCR and western blot analysis. Immunofluorescence Localization of FKHR and FKHRL1 RD cells were seeded in 4 well chamber slide and cultured in RPMI 1640 medium containing 0.5% FBS 48 hours before treatment with LY294002, or DMSO, or blank 0.5% FBS RPMI 1640 without drug. Immunofluorescence was carried out 2 hour post-treatment and follow the procedures as described before (27). Cy3- conjugated goat anti-rabbit IgG (Jackson ImmunoResearch) was used for FKHR or FKHRL1 detection. Cell nuclears were counter stained with DAPI. Images were captured on an SKY-300V spectral imager (Applied Spectral Imaging) DNA Constructs, Transfection and Selection of Clones EWS-FLI1 type I fusion cDNA was cloned into pcDNA4/TO (Invitrogen), a tetracycline inducible expression vector, and named pcDNA/TO-EF. The same EWS-FLI1 type I fusion cDNA was cloned in the anti-sense manner into pcDNA3 (Invitrogen) and named pcDNA-ASEF. All transfections were done with Effectene (Qiagen) according to the manufacturer. Twenty-four hour post-transfection, TC71 cells were split and selected in culture medium containing 150 pg/ml G418 (Invitrogen/Gibco). Two weeks after transfection, single clones were picked up and expanded. The same dose of G418 used for selection was added to the medium for maintenance of stable clones. To set up the tetracycline-regulated system (The T-Rex System, Invitrogen), RD cells were first stably transfected with pcDNA6/TR and 128 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. monoclones were selected and maintained in tetracycline-free media containing 5 pg/ml Blasticidin (Invitrogen). The inducibility of each clone was tested by transient transfection with pcDNA4/TO/LacZ and then stained for P-gal using P-gal staining kit (Invitrogen). Two out of 40 pcDNA6/TR clones were chosen for second stable transfection with pcDNA/TO-EF. Tetracycline-free media containing both 5 pg/ml Blasticidin and 400 pg/ml Zeocin (Invitrogen) was used for selecting and maintaining monoclones. Induction of EWS-FLI1 was accomplished by adding 1 pg/ml tetracycline to growth media and tested by RT-PCR and western blot. Cell Proliferation Assays For cell growth curve analysis, the same number of cells was seeded in 12 well plates and cell number was counted every 24 hour for 3 consecutive days before reaching confluence. In H -Thymidine incorporation assay, the same number of cells was seeded in 48 well plates with 0.5 ml medium. Each of un-transfected, pcDNA3 ■ i transfected, and pcDNA-ASEF transfected cells had 3 wells for measuring H - Thymidine incorporation, 1 well for measuring the background of radioactivity, and another 3 wells for cell count. Twenty-four hours after seeding, cells were counted and 1 pCi H3 -Thymidine (ICN) was then added to the medium of wells for measuring H3 activity. The wells for background measuring received no treatments. After 4 hour incubation at 37 °C, treated and untreated cells were washed three times with cold PBS and then dissolved in Solvable (NEN) 200 pl/well. For each well, 150 pi cell lysis was then transferred to the scintillation vial containing 5 ml scintillation counting solution (Fisher chemicals). H3 -Thymidine activity was measured with 129 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. scintillation counter (Beckman). After subtracting the background reading, the mean and standard deviations of the readings from triplicate wells were calculated. The distribution of cells in Go/Gi phase, S phase, and G2/M phase was determined by measuring DNA content as described before. Flow cytometric analysis was performed on FACStar (Becton Dickinson) and the cell cycle status was analyzed with Mac-cycle software. RESULTS Primary ET and RMS tissues have different expression patterns of D type cyclins and cyclin E l. We studied the gene expression profiles of 7 ET, 8 ARMS, and 5 ERMS tumors using Affymetrix® U95Av2 arrays and the relative expression level for each probe set across different samples was compared. On the U95Av2 array, there are 65 probe sets for G1 cyclins, CDKs, CDK-activating kinase (CAK, a complex formed by CDK7, cyclin H, and MAT1), CDK inhibitors, the RB protein family (RBI, RBLl/pl07, and RBL2/pl30), the E2F transcription factors (E2F1 to E2F6, TFDP1, and TFDP2), p53, MDM2, and MDM4. For each probe set, we compared the average of the relative expression value in RMS with that in ET. Of these probe sets, only those for the cyclin D1 gene (CCND1) had >2 fold higher average expression value in ET compared to RMS, while the probe sets for the cyclin D2 gene (CCND2) the cyclin D3 gene (CCND3) and the cyclin El gene (CCNE1) had >2 fold higher average expression value in RMS compared to ET (Fig. 1 A). Expression levels of the CDK inhibitor, CDKNlC/p57, were significantly high in 6 130 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RMS samples, but low in the other RMS samples and most ET samples (data not shown). Levels of proliferating cell nuclear antigen (PCNA) and MKI-67 were only slightly higher (less than 1 fold) in RMS samples than ET (data not shown). Thus, the observed differences in D type cyclin expression were not due to different proliferation rates between ET and RMS samples analyzed in this data set. Based on the expression of 10 probe sets for D type and E type cyclins, hierarchical clustering was able in every case to distinguish 7 ET from 13 RMS samples (Fig. 1 A). This differential expression pattern of G1 cyclins was more striking when the 8 ARMS samples were compared with the 7 ET samples (Fig. 1 A). In contrast, Cyclin E2 gene (CCNE2) expression was variable in both tumor types. Cyclin D1 is the major D cyclin expressed in ET, while cyclin D2 and D3 are the major D cyclins expressed in RMS. To confirm different expression patterns of G1 cyclins in ET and RMS, we assayed G1 cyclin expression in 3 primary ET tumors, 3 RMS tumors, 7 ET cell lines and 6 RMS cell lines (see Materials and Methods for fusion gene status) at mRNA levels using quantitative real time RT-PCR and protein levels using western blot. Consistent with the microarray data, ET tumors had higher cyclin D1 mRNA and protein levels, while RMS tumors had higher levels of cyclin D2 and D3 in the sample-to-sample comparisons (Fig. 1, B and Q . More striking differences in the expression of D cyclins were observed when the ET and RMS cell lines were compared (Fig. 2, A and B). Cyclin D1 expression in ET cell lines was previously reported to be variable (9) or consistently high (10). Although we also observed some variation in cyclin 131 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. D l, we found that this variation appeared to be correlated with EWS-FLI1 and EWS- ERG fusion gene expression in the ET cell lines (Fig. IB). TC135, which has the lowest EWS-FLI1 expression level in the ET lines tested, showed the lowest level of cyclin Dl but high level of cyclin D3 expression. A4573, which expresses an EWS- FLI1 type 3 fusion gene, showed similar cyclin Dl expression, but has higher cyclin D2 and D3 expression than the other ET lines (Fig. 2A and B). Compared to ET, RMS tumors and cell lines generally had higher cyclin El mRNA (Fig. IB and Fig. 2A) and proteins (Fig. 1C and Fig. 2B). Cyclin E2 appears to be the major E type cyclin expressed in RMS at the mRNA level (Fig. IB and Fig. 2A), but this was not evident at the protein level (Fig. 1C and Fig. 2B). At the protein level, cyclin E2 appeared to be high in samples with low cyclin El levels. No significant differences in the expression of CDKs (CDK2, CDK4, and CDK6) or CDK inhibitors (CDKN2A/pl6, CDKN2B/pl5, CDKN2C/pl8, CDKN2D/pl9, CDKN1A/P21, CDKN1B/P27, and CDKN1C/P57) were observed between the RMS lines and the ET lines (data not shown). We next immunoprecipitated cyclin D-CDK4 complexes with anti-CDK4 antibodies and then tested for the presence and abundance of each D type cyclin. These studies confirmed that the major D cyclins expressed in the RMS lines (Rhl8 and TC487) and ET lines (TC32 and TC466) were also the major cyclins associated with their catalytic partner, CDK4 (Fig. 2C). MEK inhibition decreases D type cyclin expression in RMS but not in ET. We next assessed whether differences in D type cyclin expression between ET 132 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and RMS were accompanied by differences in upstream mitogenic signaling pathways. In many cell types, the extracellular signal-regulated protein kinases ERK1 and ERK2 relay mitogenic signals from membrane growth factor receptors to the nucleus (28, 29). Activation of ERK1/2 has been shown to be sufficient to induce cyclin Dl transcription even in the absence of growth factors (30). Whether cyclin D2 and D3 expression is regulated by ERK1/2 is not known. Constitutive activation of ERK1/2 was observed in all RMS tumors and cell lines tested (data not shown). In contrast, ERK1/2 activities varied in ET tumors and cell lines tested and no correlation between ERK1/2 activities and cyclin Dl expression was observed in ET (data not shown), which is consistent with a previous report (15). We then blocked ERK1/2 activation in ET (TC71 and 6647) and RMS (Rhl8 and RD) cell lines with the mitogen activated protein kinase kinases (MEK1/2) inhibitor, U0126 (31, 32). ET and RMS cells were serum starved in 0.5% FBS medium before and during treatment with either U0126 or DMSO vehicle control. Significant inhibition of ERK1/2 activation (especially p44 ERK1) was observed by 4 hours and continued until at least 24 hours post-U0126 treatment in all the ET and RMS cells tested (Fig. 3^4). MEK inhibition, however, had no effect on D-type cyclin expression in ET cells, at both the RNA and the protein levels (Fig. 3, A and B). Cyclin D2 protein was virtually undetectable in ET cell lines. In contrast, MEK inhibition in RMS cells was accompanied by a significant decrease in the expression of all three D-type cyclins (Fig. 3, A and B). This was observed by 8 hours and reached maximum levels at 24 hours post-treatment (Fig. 3A). 133 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. PI3K inhibition decreases D cyclin expression in both RMS and ET cells. D-type cyclins are also regulated at the post-transcriptional level, although most work in this regard has focused on cyclin Dl regulation. Phosphatidylinositol 3- kinase (PI3K)-dependent signaling pathways have been shown to up-regulate cyclin Dl translation and down-regulate cyclin Dl degradation in response to mitogenic stimuli (33, 34). We therefore evaluated the role of the PI3K-AKT pathway in D- type cyclin regulation in ET and RMS. Again, ET and RMS cells were starved in 0.5% FBS medium before and during treatment with either the PI3K inhibitor LY294002 or DMSO vehicle control. LY294002 treatment markedly reduced AKT phosphorylation and D cyclin proteins in both ET and RMS cells (Fig. 3C). No decreases in D cyclin RNAs were observed in LY294002 treated ET cells (Fig. 3D), implying that PI3K blockade inhibited D cyclin expression at the post-transcriptional level. Again, cyclin D2 protein was virtually undetectable in ET cells. In contrast, significant decreases in all 3 D cyclin RNAs were observed in LY294002 treated RMS cells (Fig. 3D), suggesting in RMS both transcriptional and post-transcriptional regulation of D cyclin expression were likely affected by PI3K blockade. PI3K inhibition in RMS cells is associated with increased nuclear localization of FOXOla/FKHR and F0X03a/FKHRLl. AKT was shown to directly phosphorylate the FOXO forkhead transcription factors, FOXOla/FKHR, F0X03a/FKHRLl, and F0X 04 /AFX, (35, 36), which led to nuclear exclusion and inhibition of FOXO factor-mediated gene expression (27, 37). Recent publication demonstrated that above three FOXO factors repress the transcription of cyclin Dl 134 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and D2 (38, 39). This may provide clues on how D cyclins are transcriptionally regulated by PI3K-AKT signaling in RMS cell lines. Our gene expression data from primary ET and RMS tumors indicated that FKHR and FKHRL1 were expressed at significantly higher levels in RMS compared to ET (data not shown). We thus tested whether phosphorylation and nuclear localization of FOXO factors differed in RMS cells following PI3K inhibition. Compared to DMSO treated RD cells, the decrease in phosphorylation of FKHRL1 Thr32 and FKHR Thr24 was evident at 2 hour post LY294002 treatment and continued through 24 hour post LY294002 treatment when corrected for loading using beta-actin as the standard (Fig. 4A). No significant changes in phosphorylation of FKHR Ser256 and AFX serine 293 were observed, which was likely due to the serum deprived growth conditions. The sub-cellular localization of FKHR and FKHRL1 with or without LY294002 treatment was then detected with Immunofluorescence. Two patterns of FOXO localization, nuclear plus cytoplasmic and nuclear alone, were observed (Fig. 45). Compared to untreated (Blank) and DMSO treated RD cells, increased nuclear localization of FKHR and FKHRL1 was observed in RD cells 2 hours post LY294002 treatment. Such increase included increased number of cells with exclusive nuclear localization and decreased cytoplasmic staining intensity in cells with both nuclear and cytoplasmic staining (Fig. 45). Anti-sense blockade of EWS-FLI1 expression in ET reduces cyclin Dl expression but increases cyclin D3 expression. Since ET is characterized by the 135 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. presence of specific gene fusions, we next wished to determine whether EWS-ETS fusion proteins play a role in the differential usage of D-type cyclins in ET. An anti sense EWS-FLI1 type 1 fusion construct was stably transfected into the TC71 ET line. Compared with un-transfected and blank vector transfected TC71 controls, expressing anti-sense EWS-FLI1 in clone ASEF9 cells significantly decreased EWS- FLI1 protein (Fig. 5A). This was associated with decreased cyclin Dl and increased cyclin D3 expression at both RNA (data not shown) and protein levels (Fig. 5A), but despite this inhibition cyclin Dl remained the dominant D cyclin. Expression of cyclin D2 remained low. No significant alterations in cyclin El expression were observed (Fig. 5A). No other significant alterations in cell cycle related proteins were detected. To test the effects of EWS-FLI1 inhibition on cell proliferation, we assessed cell proliferation by cell counting and observed a significant decrease in the number of ASEF9 cells compared to controls (Fig. 5B). To determine the proliferation ratios, S phase DNA synthesis activity was measured by H -Thymidine incorporation. Compared to TC71 controls, H3 -Thymidine incorporation was 2 fold less in ASEF9 cells by 28 hours after seeding (Fig. 5C). Consistent with this, a 50% increase in the percentage of ASEF9 cells in Go/Gi phase was detected by flow cytometry (Fig. 5£>). Similar results were observed in additional clones stably expressing anti-sense EWS-FLI1 (data not shown). These data not only confirm that EWS-FLI1 is essential for ET cell proliferation, but also that, expression of this chimeric oncoprotein specifically correlates with differential expression of cyclin Dl and D3. 136 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. EWS-FLI1 increases cyclin D l but decreases cyclin D3 expression in RMS. To further analyze the roles of EWS-FLI1 on D-type cyclin expression and the effect of ERK1/2 activation in this process we established a tetracycline controlled EWS-FLI1 expression system in the RMS cell line, RD (RD/trexEF). ERK1/2 phosphorylation and the expression of D cyclins were followed at different time points after EWS-FLI1 induction. As shown in figure 6A and 6B, expression of EWS-FLI1 protein was first detectable 6 hours post induction and levels peaked by 12 hours. Significant increases in cyclin Dl protein were first observed at 6 hours post induction and continued to increase at subsequent time points. Although no changes in cyclin D2 were observed, expression of cyclin D3 was markedly reduced after induction of EWS-FLI1. In fact, cyclin Dl replaced cyclin D3 as the major D cyclin expressed in RD after induction of EWS-FLI1. This reversal of D-type cyclin expression following EWS-FLI1 induction was also observed at the RNA level by real time RT-PCR (data not shown). ERK1/2 activities were only transiently up- regulated at early time points, likely due to changing of medium at 0 hour, and then decreased as EWS-FLI1 protein began to accumulate (Fig. 6, A and B). Thus expression changes in cyclin Dl and D3 following EWS-FLI1 induction appeared to be independent of ERK1/2 activation. These data corroborate those of the anti-sense studies indicating that EWS-FLI1 specifically regulates cyclin Dl and D3 usage in tumor cells. We also assessed whether EWS-FLI1 induction and its effects on cyclin Dl and D3 expression altered the proliferation of RD/trexEF cells by comparing their 137 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cell cycle distribution with flow cytometry. We found that RD cells became arrested in G1 phase 24 hours after EWS-FLI1 induction (Fig. 6C). Similar results were obtained with additional RD/trexEF clones (data not shown). Although EWS-FLI1 protein is required for ET cell proliferation as suggested by the anti-sense studies, it alone is not sufficient to promote cell proliferation. DISCUSSION To compare the molecular mechanisms involved in regulating the proliferation of ET and RMS tumor cells, we studied the expression profile of genes involved in cell cycle G1 progression and identified different expression patterns of D type cyclins in these two childhood tumors types. Both primary tumor and tumor derived cell line studies demonstrated that cyclin Dl was the major D type cyclin in ET, while cyclin D2 and D3 predominated in RMS. These observations were confirmed at both the RNA and protein levels. In addition, we found that while signaling through both RAS-ERK1/2 and PI3K-AKT cascades regulated D type cyclin expression in RMS, D type cyclin expression in ET appeared to be regulated at the post-transcriptional level by PI3K-AKT. Our data further indicate that expression of the EWS-FLI1 fusion protein can influence the levels of cyclin Dl and D3 in both ET and RMS cellular backgrounds and that this is independent of ERK1/2 activation. Most studies of D-type cyclin regulation have focused on cyclin Dl. In our study, blocking sustained ERK1/2 activation by MEK1/2 inhibition abolished the 138 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. expression of all three D cyclins in RMS cell lines but not in ET lines. Thus in RMS RAS-ERK1/2 signaling not only regulates cyclin Dl expression but also that of cyclin D2 and D3. Sustained RAS-ERK1/2 signaling is thought to induce transcription of the cyclin Dl promoter through induction of specific transcription factors (40, 41). Although D cyclin promoters have marked differences in their regulatory elements, consensus binding sites for a number of transcription factors implicated in transcriptional regulation of cyclin Dl expression, including activator protein-1 (AP-1) and Spl, are also present on cyclin D2 and D3 promoters (21, 22, 41). It is possible that MEK1/2 inhibition in our studies was acting to block the function of these or other transcription factors in RMS cells, leading to the observed decreases in expression of all three D cyclins. To elucidate how the different cellular backgrounds of RMS and ET might contribute to the differential responses to MEK1/2 inhibition, we reviewed the Affymetrix gene expression profiles of nuclear factors in ET and RMS primary tumor samples. This demonstrated that Id2 (inhibitor of DNA binding 2), a helix-loop-helix (HLH) protein, was much more highly expressed in ET compared to RMS (data not shown). In support of this, Id2 was recently identified as a downstream target of the EWS-ETS fusion proteins and was over-expressed in ET tumor and cell lines (42, 43). Deregulated expression of Id proteins (Id 1-3) has been reported in several human tumor types, and has been implicated in the regulation of tumor growth, angiogenesis, invasiveness and metastasis (44, 45). Although not directly binding to DNA, Id2 has been shown to block RAS-ERK1/2 signaling to SRE (serum respond element) by down-regulating 139 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the transcriptional activity mediated by the ternary complex factors (46). It is possible that high level of Id2 in ET blocks regulation of cyclin D expression by RAS-ERK1/2 signaling. The PI3K-AKT pathway is also known to regulate cyclin D1 expression. PI3K activation of p70 S6 kinase increases cyclin D1 translation (33). Moreover, glycogen synthase kinase 30 (GSK30) phosphorylates cyclin D1 and promotes its proteasomal degradation (34, 47), and AKT inhibits this function by phosphorylating GSK30 at serine 9 (48, 49). In our study, sustained AKT activation was observed in all the ET and RMS samples tested and PI3K inhibition reduced expression of all three D cyclins in both ET and RMS cells. D cyclin levels in RMS therefore appear to represent a functional balance between signaling through the RAS-ERK1/2 and PI3K-AKT pathways. Although the mechanism(s) by which PI3K-AKT signaling regulates D type cyclin expression in these tumors remains unknown, there appear to be distinct differences in this regard between ET and RMS. In ET, PI3K inhibition decreased D cyclin expression at the post-transcriptional level, while in RMS such decreases appear to be at both transcriptional and post-transcriptional levels. When we tested the roles of FKHR and FKHRL1 in regulating D cyclin transcription in RMS lines, we found decreased phosphorylation and subsequent increased FKHR and FKHRL1 nuclear localization shortly after blocking PI3K-AKT signaling in the RD RMS line. Although only one of the two AKT phosphorylation sites (e.g., Thr24) on FKHR was blocked by LY294002 treatment in serum starved RD cells, such blockade is sufficient to increase the nuclear localization of FKHR. Both 140 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. FKHR/FOXOla and FKHRLl/F0X03a have been shown to repress cyclin D1 and D2 transcription (38, 39). However, the common FOXO DNA binding element, the insulin response sequences (IRS), is not required for the repression of CCND1 and CCND2 promoter activities (39). Whether FKHR and FKHRL1 repress D cyclin transcription indirectly by interacting with other transcription factors or directly by binding to new DNA response elements is not clear. In addition to cyclin D1 and D2, we found blocking PI3K-AKT signaling also appeared to decrease cyclin D3 RNA. We are currently attempting to block FKHR and FKHRL1 expression using RNAi and will then follow the transcription of all three D cyclins following this blockade in RMS lines. Accumulating data has illustrated a correlation between the expression of EWS-FLI1 and cyclin D1 (10, 50, 51), but whether EWS-FLI1 directly or indirectly regulates cyclin D1 expression is not well documented. As a transcription factor, EWS-FLI1 binds DNA in a sequence specific manner (52). Potential EWS-FLI1 binding sites can be identified in D cyclin promoters (21, 22). A recent paper demonstrated that EWS-FLI1 directly binds to the cyclin D1 promoter region (42), indicating that cyclin D1 may be a direct target of this fusion protein. Indeed, when we induced EWS-FLI1 expression in RD cells both cyclin D1 and D3 were among the early response genes following EWS-FLI1 induction. Our unpublished data also showed that ectopically expressing EWS-FLI1 in RD cells could transactivate the cyclin D1 promoter and that this transactivation required the DNA binding ability of EWS-FLI1. Whether EWS-FLI1 directly regulates cyclin D3 expression remains to 141 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. be determined. If so, it is possible that interactions between EWS-FLI1 with other nuclear factors may determine its ability to up-regulate cyclin D1 while down- regulating cyclin D3 transcription in ET. Forced expression of EWS-FLI1 was previously shown to up-regulate ERK1/2 activity in NIH3T3 cells (53), raising the possibility that EWS-FLI1 can indirectly regulate D cyclin expression through ERK1/2. However, our data and that of others (15) indicate that RAS-ERK1/2 activation may not be essential for D cyclin expression in ET. Although the mechanism by which EWS-FLI1 activates ERK1/2 is not clear, such regulation may depend on cellular background (i.e. in ET versus RMS). No increases in ERK1/2 activation were observed when we induced EWS- FLI1 expression in the RMS line, RD. Instead, with the accumulation of EWS-FLI1 protein, ERK1/2 activation in RD-TREX cells actually declined. If EWS-FLI1 modulates D cyclin expression through ERK1/2 signaling, it would be difficult to explain why induction of EWS-FLI1 in RD cells increased cyclin D l, decreased cyclin D3, and had no effects on cyclin D2 expression. Expression of cyclin Dl in ET therefore appears to be transcriptionally regulated by EWS-FLI1 and post- transcriptionally regulated by the PI3K-AKT signaling. Ectopic expression of EWS-FLI1 in primary human fibroblasts and primary mouse embryo fibroblasts was previously shown to induce p53 or pl6INK4A dependent growth arrest (54, 55). The early passage (e.g., p i8) RD line used in our study has mis-sense mutations in p53, but does express pl6INK4A (data not 142 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. shown)*. To elucidate the role of EWS-FLI1 in cell proliferation we choose TC71, an ET line with a nonsense mutation in the p53 gene and lacking CDKN2A/pl6 expression (J. Zhang et al, unpublished data), in our anti-sense study. We found that blocking EWS-FLI1 expression in TC71 cells could still lead to cell cycle G1 arrest (see Fig. 5D). Therefore it appears that while EWS-FLI1 itself may not be sufficient to accelerate cell proliferation, it is required for the proliferation of ET cells in which pro-apoptotic or growth arrest pathways have been inactivated. Although the underlying mechanisms by which EWS-FLI1 mediates its proliferative effects are largely unknown, it is likely that they involve the up-regulation of positive cell cycle regulators such as cyclin D l. In conclusion, our results indicate that different D type cyclins control proliferation of ET and RMS cells. This selective usage of D type cyclins is due to, at least in part, the different downstream effects of common mitogenic signaling pathways as well as the expression of the EWS-FLI1 fusion gene in ET. Further studies will delineate the detailed mechanisms regulating D cyclin expressions in these two common childhood sarcomas and highlight targets that could be used for developing tumor specific therapeutic agents based on this information. * O ther later passages o f RD have been reported to lack p l6IN K 4A [56] 143 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS We are grateful to Dr. Michael J. Anderson for reviewing the manuscript and to Carol Peebles for assistance in this project. We thank Betty Schaub, Nilmini Waidyaratne, and Violette Shahbazian for their technical support. This work was supported in part by a Director’s Challenge grant from the National Cancer Institute (TJT, CA88199-01). This work was also supported by the Las Madrinas Endowment for Molecular Pathology (TJT) and the Fannie Rippel Foundation (PHS). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. REFERENCES: 1. Amdt, C. A. and Crist, W. M. Common musculoskeletal tumors of childhood and adolescence. N Engl J Med 1999; 341/ 342-352. 2. Franchi, A., Pasquinelli, G., Cenacchi, G., Della Rocca, C., Gambini, C., Bisceglia, M., Martinelli, G. N., and Santucci, M. Immunohistochemical and ultrastructural investigation of neural differentiation in Ewing sarcoma/PNET of bone and soft tissues. Ultrastruct Pathol 2001; 25/ 219-225. 3. Arvand, A. and Denny, C. T. Biology of EWS/ETS fusions in Ewing's family tumors. Oncogene 2001; 20/ 5747-5754. 4. Delattre, O., Zucman, J., Plougastel, B., Desmaze, C., Melot, T., Peter, M., Kovar, H., Joubert, I., de Jong, P., Rouleau, G., and et al. Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature 1992; 359/ 162-165. 5. Sorensen, P. H., Lessnick, S. L., Lopez-Terrada, D., Liu, X. F., Triche, T. J., and Denny, C. T. A second Ewing's sarcoma translocation, t(21;22), fuses the EWS gene to another ETS-family transcription factor, ERG. Nature Genetics 1994; 6/ 146-151. 6. Merlino, G. and Helman, L. J. Rhabdomyosarcoma— working out the pathways. Oncogene 1999; 18/ 5340-5348. 7. Barr, F. G. Gene fusions involving PAX and FOX family members in alveolar rhabdomyosarcoma. Oncogene 2001; 20/ 5736-5746. 8. Kovar, H., Auinger, A., Jug, G., Aryee, D., Zoubek, A., Salzer-Kuntschik, M., and Gadner, H. Narrow spectrum of infrequent p53 mutations and absence of MDM2 amplification in Ewing tumours. Oncogene 1993; 8/ 2683-2690. 9. Kovar, H., Jug, G., Aryee, D. N., Zoubek, A., Ambros, P., Gruber, B., Windhager, R., and Gadner, H. Among genes involved in the RB dependent cell cycle regulatory cascade, the p i6 tumor suppressor gene is frequently lost in the Ewing family of tumors. Oncogene 1997; 15/ 2225-2232. 10. Dauphinot, L., De Oliveira, C., Melot, T., Sevenet, N., Thomas, V., Weissman, B. E., and Delattre, O. Analysis of the expression of cell cycle regulators in Ewing cell lines: EWS-FLI-1 modulates p57KIP2and c-Myc expression. Oncogene 2001; 20/ 3258-3265. 11. Felix, C. A., Kappel, C. C., Mitsudomi, T., Nau, M. M., Tsokos, M., Crouch, G. D., Nisen, P. D., Winick, N. J., and Helman, L. J. Frequency and diversity of p53 mutations in childhood rhabdomyosarcoma. Cancer Res 1992; 52/ 2243-2247. 12. Mulligan, L. M., Matlashewski, G. J., Scrable, H. J., and Cavenee, W. K. Mechanisms o f p53 loss in human sarcomas. Proc Natl Acad Sci U S A 1990; 87/ 5863-5867. 13. Iolascon, A., Faienza, M. F., Coppola, B., Rosolen, A., Basso, G., Della Ragione, F., and Schettini, F. Analysis of cyclin-dependent kinase inhibitor 145 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. genes (CDKN2A, CDKN2B, and CDKN2C) in childhood rhabdomyosarcoma. Genes Chromosomes Cancer 1996; 15: 217-222. 14. De Chiara, A., T'Ang, A., and Triche, T. J. Expression of the retinoblastoma susceptibility gene in childhood rhabdomyosarcomas. J Natl Cancer Inst 1993; 85: 152-157. 15. Lawlor, E. R., Scheel, C., Irving, J., and Sorensen, P. H. Anchorage- independent multi-cellular spheroids as an in vitro model of growth signaling in Ewing tumors. Oncogene 2002; 21: 307-318. 16. Khan, J., Wei, J. S., Ringner, M., Saal, L. H., Ladanyi, M., Westermann, F., Berthold, F., Schwab, M., Antonescu, C. R., Peterson, C., and Meltzer, P. S. Classification and diagnostic prediction of cancers using gene expression profiling and artificial neural networks. Nat Med 2001; 7: 673-679. 17. Suzuki, R., Kuroda, H., Komatsu, H., Hosokawa, Y., Kagami, Y., Ogura, M., Nakamura, S., Kodera, Y., Morishima, Y., Ueda, R., and Seto, M. Selective usage of D-type cyclins in lymphoid malignancies. Leukemia 1999; 13: 1335-1342. 18. Sherr, C. J. Mammalian G1 cyclins. Cell 1993; 73: 1059-1065. 19. Hunter, T. and Pines, J. Cyclins and cancer. II: Cyclin D and CDK inhibitors come of age. Cell 1994; 79: 573-582. 20. Sherr, C. J. Cancer cell cycles. Science 1996; 274: 1672-1677. 21. Herber, B., Truss, M., Beato, M., and Muller, R. Inducible regulatory elements in the human cyclin Dl promoter. Oncogene 1994; 9: 1295-1304. 22. Brooks, A. R., Shiftman, D., Chan, C. S., Brooks, E. E., and Milner, P. G. Functional analysis of the human cyclin D2 and cyclin D3 promoters. J Biol Chem 1996; 271: 9090-9099. 23. Matsushime, H., Roussel, M. F., Ashmun, R. A., and Sherr, C. J. Colony- stimulating factor 1 regulates novel cyclins during the G1 phase of the cell cycle. Cell 1991; 65: 701-713. 24. Inaba, T., Matsushime, H., Valentine, M., Roussel, M. F., Sherr, C. J., and Look, A. T. Genomic organization, chromosomal localization, and independent expression of human cyclin D genes. Genomics 1992; 13: 565- 574. 25. Tam, S. W., Theodoras, A. M., Shay, J. W., Draetta, G. F., and Pagano, M. Differential expression and regulation of Cyclin Dl protein in normal and tumor human cells: association with Cdk4 is required for Cyclin Dl function in G1 progression. Oncogene 1994; 9: 2663-2674. 26. Wu, L., Chen, P., Shum, C. H., Chen, C., Barsky, L. W., Weinberg, K. I., Jong, A., and Triche, T. J. MAT 1-modulated CAK activity regulates cell cycle G(l) exit. Mol Cell Biol 2001; 21: 260-270. 27. Biggs, W. H., 3rd, Meisenhelder, J., Hunter, T., Cavenee, W. K., and Arden, K. C. Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1. Proc Natl Acad Sci U S A 1999; 96: 7421-7426. 146 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 28. Marshall, C. J. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 1995; 80: 179-185. 29. Cobb, M. H. and Goldsmith, E. J. How MAP kinases are regulated. J Biol Chem 1995; 270: 14843-14846. 30. Lavoie, J. N., L'Allemain, G., Brunet, A., Muller, R., and Pouyssegur, J. Cyclin Dl expression is regulated positively by the p42/p44MAPK and negatively by the p38/HOGMAPK pathway. J Biol Chem 1996; 271: 20608- 20616. 31. Favata, M. F., Horiuchi, K. Y., Manos, E. J., Daulerio, A. J., Stradley, D. A., Feeser, W. S., Van Dyk, D. E., Pitts, W. J., Earl, R. A., Hobbs, F., Copeland, R. A., Magolda, R. L., Scherle, P. A., and Trzaskos, J. M. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem 1998; 273: 18623-18632. 32. Davies, S. P., Reddy, H., Caivano, M., and Cohen, P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 2000; 351: 95-105. 33. Muise-Helmericks, R. C., Grimes, H. L., Bellacosa, A., Malstrom, S. E., Tsichlis, P. N., and Rosen, N. Cyclin D expression is controlled post- transcriptionally via a phosphatidylinositol 3-kinase/Akt-dependent pathway. J Biol Chem 1998; 273: 29864-29872. 34. Diehl, J. A., Cheng, M., Roussel, M. F., and Sherr, C. J. Glycogen synthase kinase-3beta regulates cyclin Dl proteolysis and subcellular localization. Genes Dev 1998; 12: 3499-3511. 35. Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., Anderson, M. J., Arden, K. C., Blenis, J., and Greenberg, M. E. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 1999; 96: 857-868. 36. Kops, G. J., de Ruiter, N. D., De Vries-Smits, A. M., Powell, D. R., Bos, J. L., and Burgering, B. M. Direct control of the Forkhead transcription factor AFX by protein kinase B. Nature 1999; 398: 630-634. 37. Brownawell, A. M., Kops, G. J., Macara, I. G., and Burgering, B. M. Inhibition of nuclear import by protein kinase B (Akt) regulates the subcellular distribution and activity of the forkhead transcription factor AFX. Mol Cell Biol 2001; 21: 3534-3546. 38. Schmidt, M., Fernandez de Mattos, S., van der Horst, A., Klompmaker, R., Kops, G. J., Lam, E. W., Burgering, B. M., and Medema, R. H. Cell cycle inhibition by FoxO forkhead transcription factors involves downregulation of cyclin D. Mol Cell Biol 2002; 22: 7842-7852. 39. Ramaswamy, S., Nakamura, N., Sansal, I., Bergeron, L., and Sellers, W. R. A novel mechanism of gene regulation and tumor suppression by the transcription factor FKHR. Cancer Cell 2002; 2: 81-91. 147 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 40. Murphy, L. 0., Smith, S., Chen, R. H., Fingar, D. C., and Blenis, J. Molecular interpretation of ERK signal duration by immediate early gene products. Nat Cell Biol 2002; 4: 556-564. 41. Roovers, K. and Assoian, R. K. Integrating the MAP kinase signal into the G1 phase cell cycle machinery. Bioessays 2000; 22: 818-826. 42. Fukuma, M., Okita, H., Hata, J., and Umezawa, A. Upregulation of Id2, an oncogenic helix-loop-helix protein, is mediated by the chimeric EWS/ets protein in Ewing sarcoma. Oncogene 2003; 22: 1-9. 43. Nishimori, H., Sasaki, Y., Yoshida, K., Irifune, H., Zembutsu, H., Tanaka, T., Aoyama, T., Hosaka, T., Kawaguchi, S., Wada, T., Hata, J., Toguchida, J., Nakamura, Y., and Tokino, T. The Id2 gene is a novel target of transcriptional activation by EWS-ETS fusion proteins in Ewing family tumors. Oncogene 2002; 21: 8302-8309. 44. Lasorella, A., Uo, T., and Iavarone, A. Id proteins at the cross-road of development and cancer. Oncogene 2001; 20: 8326-8333. 45. Norton, J. D. ID helix-loop-helix proteins in cell growth, differentiation and tumorigenesis. J Cell Sci 2000; 113 ( Pt 22): 3897-3905. 46. Yates, P. R., Atherton, G. T., Deed, R. W., Norton, J. D., and Sharrocks, A. D. Id helix-loop-helix proteins inhibit nucleoprotein complex formation by the TCF ETS-domain transcription factors. Embo J 1999; 18: 968-976. 47. Shao, J., Sheng, H., DuBois, R. N., and Beauchamp, R. D. Oncogenic Ras- mediated cell growth arrest and apoptosis are associated with increased ubiquitin-dependent cyclin Dl degradation. J Biol Chem 2000; 275: 22916- 22924. 48. Cross, D. A., Alessi, D. R., Cohen, P., Andjelkovich, M., and Hemmings, B. A. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 1995; 378: 785-789. 49. van Weeren, P. C., de Bruyn, K. M., de Vries-Smits, A. M., van Lint, J., and Burgering, B. M. Essential role for protein kinase B (PKB) in insulin-induced glycogen synthase kinase 3 inactivation. Characterization of dominant- negative mutant of PKB. J Biol Chem 1998; 273: 13150-13156. 50. Matsumoto, Y., Tanaka, K., Nakatani, F., Matsunobu, T., Matsuda, S., and Iwamoto, Y. Downregulation and forced expression of EWS-Flil fusion gene results in changes in the expression of G(l)regulatory genes. Br J Cancer 2001; 84: 768-775. 51. Eliazer, S., Spencer, J., Ye, D., Olson, E., and Ilaria, R. L., Jr. Alteration of Mesodermal Cell Differentiation by EWS/FLI-1, the Oncogene Implicated in Ewing's Sarcoma. Mol Cell Biol 2003; 23: 482-492. 52. Mao, X., Miesfeldt, S., Yang, H., Leiden, J. M., and Thompson, C. B. The FLI-1 and chimeric EWS-FLI-1 oncoproteins display similar DNA binding specificities. Journal of Biological Chemistry 1994; 269: 18216-18222. 53. Silvany, R. E., Eliazer, S., Wolff, N. C., and Ilaria, R. L., Jr. Interference with the constitutive activation of ERK1 and ERK2 impairs EWS/FLI-1- dependent transformation. Oncogene 2000; 19: 4523-4530. 148 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 54. Lessnick, S. L., Dacwag, C. S., and Golub, T. R. The Ewing's sarcoma oncoprotein EWS/FLI induces a p53-dependent growth arrest in primary human fibroblasts. Cancer Cell 2002; 1: 393-401. 55. Deneen, B. and Denny, C. T. Loss of pi 6 pathways stabilizes EWS/FLI1 expression and complements EWS/FLI 1 mediated transformation. Oncogene 2001;20:6731-6741. 56. Urashima, M., Teoh, G., Akiyama, M., Yuza, Y., Anderson, K. C., and Maekawa, K. Restoration of pl6INK4A protein induces myogenic differentiation in RD rhabdomyosarcoma cells. Br J Cancer 1999; 79: 1032- 1036. 149 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. FIGURE LEGENDS Fig. 1. Expression of D type cyclins and E type cyclins in ET and RMS primary tumor samples. A, hierarchical clustering of primary tumors samples and G1 cyclins. Each row represents the relative expression level of each probe set across different samples. Its gene name, Genbank locus, and the ratio of average expression levels between ET and RMS are indicated on the right. Each column represents an individual sample. The blue line underneath the first 3 columns indicates the triplicates of the same ET sample. The orange lines underneath other columns indicate the 8 ARMS samples. The relative expression level is represented by the red (high expression) and green (low expression) pseudo-color and the “trust” of the data is indicated by the saturation of the color, where unsaturated color represents low trust and saturated color represents high trust. In the scale below, the horizontal axis represents the scale for normalized expression value and the vertical axis is the “trust” scale. B, relative expression of D type cyclin and E type cyclin mRNAs in ET (ET98, ET96, and ETOO), ERMS (Re94), and ARMS (Ra96, and Ra97) primary tumor samples. Real time quantitative RT-PCR was performed as described in “Materials and Methods”. For each sample, the average and the standard deviation of the Cyclin/p-Actin ratio from at least three independent PCR reactions is shown. C, western blots detecting D type cyclins and E type cyclins in ET and RMS tumors. 0- Actin is shown as the loading control. Fig. 2. Expression of D type cyclins and E type cyclins in 7 ET (6647, RDES, TC135, TC71, TC32, TTC466, and A4573) and 6 RMS (RD, TC442, Rhl8, Rh28, 150 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Rh30, and TC487) cell lines. A, relative expression of D type cyclin and E type cyclin mRNAs in ET and RMS cell lines. For each sample, the average and the standard deviation of the Cyclin/p-Actin ratio from at least three independent real time quantitative RT-PCR reactions are shown. B, Western Blots detecting expression of cyclin D, cyclin E, and fusion protein EWS-FLI1 or EWS-ERG in ET and RMS lines. EWS-FLI1 type III fusion (A4573) and type II fusion (RDES and 6647) have larger fusion proteins than the type I fusion, P-actin is shown as the loading control. C, Western blots detecting D type cyclin protein levels in cell lysates immunoprecipitated with anti-CDK4. Fig. 3. MEK1/2 inhibition blocked ERK1/2 activation and decreased D type cyclin expression in serum starved RMS cells but not in serum starved ET cells, whereas PI3K inhibition blocked AKT activation and decreased cyclin D expression in both serum starved RMS cells and serum starved ET cells. A, western blots detecting ERK1/2 phosphorylation at Thr202/Tyr204 (p-ERK) and the expression of D type cyclins at different hours following U0126 (U) or DMSO (D) treatment of 6647 and RD cells in 0.5% FBS culture medium. Total ERK1/2 (t-ERK) is shown as the loading control. B, relative expression of D cyclin mRNAs in ET (6647 and TC71) and RMS (Rhl8 and RD) cell lines following 12 hour treatment with U0126, DMSO, or drug-free culture medium with 0.5% FBS (Blank). The mean and standard deviation of Cyclin D/p-Actin ratio from at least three independent real time quantitative RT-PCR reactions are shown. C, western blots detecting AKT phosphorylation at Ser473 (p-AKT) and the expression of D type cyclins at different 151 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. hours following LY294002 (LY) or DMSO (D) treatment of 6647 and Rhl8 cells in 0.5% FBS culture medium. Total AKT (t-AKT) is shown as the loading control. D, expression of D cyclin RNAs in ET (6647 and TC71) and RMS (Rhl8, Rh28, and Rh30) cell lines following 12 hour treatment with LY294002, DMSO, or drug-free culture medium with 0.5% FBS (Blank). The mean and standard deviation of at least three independent real time quantitative RT-PCR reactions are shown. Fig. 4. Decreased FKHR and FKHRL1 phosphorylations and increased nuclear localizations of FKHR and FKHRL1 in RD cells following PI3K blockade. A, western blots detecting FKHR phosphorylation at Thr32 (pFKHRt) and Ser256 (pFKHRs), FKHRL1 phosphorylation at Thr32 (pFKHRLlt), and AFX phosphorylation at Seri 93 (pAFXs). B, immunofluorescence detecting sub-cellular localization of Cy3 labeled FKHR (C-F) and Cy3 labeled FKHRL1 (C-FL) in untreated (Blank), DMSO treated, or LY294002 treated RD cells. The combined images of DAPI nuclear stain with Cy3 FKHR (C-F & D) or Cy3 FKHRL1 (C-FL & D) are also shown. Fig. 5. Inhibition of EWS-FLI1 expression in TC71 cells results in decreased cyclin Dl and increased cyclinD3 levels, with cell cycle G1 arrest. A, comparison of EWS- FLI1, cyclin D l, cyclin D2, cyclin D3 and cyclin El protein levels in un-transfected (Blank), pcDNA3 vector transfected (Vector) and pcDNA-ASEF transfected monoclone # 9 (ASEF9). For detection of cyclin D2 expression, 3 times more total • > cellular proteins were used. B, growth curve analysis. C, H -Thymidine 152 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. incorporation assay. D, FACS analysis of cell cycle distribution in controls (Blank and Vector) and ASEF9 cells. Fig. 6. Inducible expression of EWS-FLI1 in RD/trexEF clone 7. A, western blots detecting EWS-FLI1 protein, G1 cyclins and ERK1/2 phosphorylation at Thr202/Tyr204 (p-ERK) at different hours following treatment with either ethanol vehicle control (C) or 1 pg/ml tetracycline (T). Total ERK (t-ERK) was used as the loading control. B, western blot band intensities in A were quantitated with FluorChem 8900. The intensity ratios of EWS-FLIl/t-ERK(EF/ERK), cyclin Dl/t- ERK(D1/ERK), cyclin D3/t-ERK(D3/ERK), phospho- p44ERK/p44ERK(pERK 1 /ERK 1), phospho-p42ERK/p42ERK(pERK2/ERK2) in ethanol control (C) or tetracycline (T) treated RD /trexEF clone 7 are plotted. C, FACS analysis comparing cell cycle distribution in un-treated RD cells (Blank), ethanol treated (ETOH-c), and RD cells at 24 hour post-induction with tetracycline (T-induced). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 1 RMS G ene Name GenBank Locus Fold Change ET/RMS RMS/ET II ■ 1 1 R l CCND1 M64349 1 5 .5 0.0646 i i" m CCND1 M73554 1 0 .5 0.0949 j t 1 i CCND1 X59798 1 2 .6 0.0793 n •* \r » I CCND2 X68452 0.474 2 .1 1 •no stii Ht j CCND2 D13639 0.397 2 .5 2 t ' 4 a k ita i CCND3 M92287 0.369 2 .7 1 n K . U g CCND3 M92287 0.483 2 .0 7 — 1 CCNE1 M73812 0.282 3 .5 5 • i i h i h i i w CCNE1 M74093 0.142 7 .0 4 i CCNE2 AF091433 0.559 1 .7 9 0 0.5 1 2 3 4 5 B 3.5 3 2.5 2 1.5 1 0.5 0 □ Cyclin Dl/Actin ■ Cyclin D2/Actin E S I Cyclin D3/Actin ET98 ET96 ET00 Re94 Ra96 Ra97 i| t l I ET98 ET96 ETOO Re94 Ra96 Ra97 3.5 □ Cyclin El/Actin E3 Cyclin E2/Actin 3 2.5 2 1.5 1 0.5 0 ET98 ET96 ETOO Re94 Ra96 Ra97 Cyclin Dl Cyclin D2 Cyclin D3 Cyclin El Cyclin E2 B-Actin Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2 3.5 3 2.5 2 1.5 1 0.5 0 □ Cyclin D1/Actin ■ Cyclin D2/Actin ■ Cyclin D3/Actin kill J < /) « r- LU « f r Q O B O ' B •- cm to c n O C M CO CO 5 C M ■ M - J = - C p C C c c . 3.5 3 - 2.5 2 - 1.5- 1 - 0.5 0 □ Cyclin E1/Actir ■ Cyclin E2/Actir I j 1 \ S (/) Ifl r N t} - h i c o r - co to ^ t - o O C O § P H H CD CO < d r - 3 | h * < C N J CO 00 o cc a: B r/s V O c o n m ^ w ^ ^ 3 P u u u u s 'O 2 E - H H ^ EWS-FLIl/ERG m u m * * Cyclin Dl CyclinDl Cyclin D2 Cyclin D2 Cyclin D3 Cyclin D3 * * m m Cyclin El Cyclin E2 B-actin ^ ^ Rhl8 TC487 TTC466 TC32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3 4hr 8hr 12hr 24hr 6647 p-ERK t-ERK Cyclin Dl Cyclin D3 R D D U D U D U D U ess* •as* 4hr 8hr 12hr 24hr D U D U D U D U n ni? if yum^ p-cKK „ t-ERK |s = ' S = ' ^rz n Cyclin Dl Cyclin D2 Cyclin D3 B 7 - •S 6 ‘ « 5 - 3 4 - 5 3 - ■ M 2 - £ 6 1 - 0 * □ Blank E S I D M S O ■ U 0 1 2 6 1 - I °’ 8 ^ 0.6 - C 4 l 0 4 § 0.2 - o - □ Blank E3DMSO ■ UOI26 . ns*. 2.5 - a 2 ’ < 1.5 - cn 9 i - i s l 6647 TC71 Rhl8 6647 TC71 Rhl8 □ Blank ^ DMSO ■ U0126 6647 TC71 Rhl8 { { A n _2hr _4hr 8hr 12hr 24hr D L Y D L Y D L Y D L Y D L Y p-AKT t-AKT Cyclin Dl Cyclin D3 2hr _4hr _ 8 h r_ L 2 h r_ 2 4 h r DLY D L Y D L Y D L Y D L Y p-AKT t-AKT Cyclin Dl Cyclin D2 Cyclin D3 -«• «* i □ Blank ESJDMSO "I LY294002 6647 TC71 Rhl8 1 I 0 '8 « ! 0.6 fS | 0.4 u & 0.2 0 □ Blank S D M S O ■ LY294002 , rK a h ., 6647 TC71 Rhl8 RD □ Blank E S I DM SO ■ LY294002 £fll 6647 TC71 Rhl8 RD 156 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 4 A 2hr 4hr 8hr 12hr 24hr ilf D LY D LY D LY D LY D LY H it - • p ^ s IIIII1I111S B-Actin B C-F C-F & D C-FL C-FL & D Blank DMSO LY294002 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5 B Blank Vector ASEF9 Cyclin Dl Cyclin D2 Cyclin D3 Cyclin El ft-Actin 14 Blank 12 8 6 4 2 0 0 1 2 3 Day O 6000 O 2000 Blank %G0/G1 = 38.1 %G2/M =21.7 = 40.1 Vector %G0/G1 = 42.2 %G2/M = 18.3 %S = 39.3 ASEF9 %G0/G1 = 62.5 %G2/M = 12.7 %S = 24.6 158 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 6 0 hr ^ S h r EWS-FLI1 Cyclin Dl Cyclin D2 Cyclin D3 Cyclin El p-ERK t-ERK B f i i i w » in iiu ir : i a t i f i p Blank %G0/G1= 41.5 %G2/M = 26.5 %S = 32.0 ETOH-c %G0/G1= 41.6 %G2/M = 21.5 %S = 36.8 T-induced %G0/G1= 75.3 %G2/M = 17.0 %S = 7.7 0.6 0 .4 0.2 18hr Ohr 3hr 6hr 12hr -o- EF/E R K -C ♦ E F /E R K -T •a- D 3/E R K -C - a- D 3/ER K -T 0.4 0.2 1 8 h r Ohr 3hr 6 h r 1 2 h r -O -p E R K l/E R K l-C - ♦ -p E R K l/E R K l-T " O pERK2/ERK2-C -* ■ pERK2/ERK2-T 0.5 Ohr 3hr 6hr 12hr 18hr 159 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Appendix 2 Lack of Interferon Response in Animals to Naked siRNAs (Manuscript has been accepted by Nature Biotechnology) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LACK OF INTERFERON RESPONSE IN ANIMALS TO NAKED SIRNAS Jeremy D. Heidel1 , Siwen Hu2, Xian Fang Liu2 , Timothy J. Triche2, and Mark E. Davis1 1 Chemical Engineering, California Institute of Technology, Pasadena, California 91125. 2 Department of Pathology, Children’s Hospital Los Angeles, CA 90027. Correspondence should be addressed to M.E.D. (mdavis@cheme.caltech.edu) RNA interference (RNAi) is rapidly becoming the method of choice for the elucidation of gene function and the identification of drug targets. As with other oligonucleotide-based strategies, RNAi is envisioned to ultimately be useful as a human therapeutic. Unlike previous nucleic acid therapeutics, small interfering RNAs (siRNAs) have the potential to elicit immune responses via interactions with Toll-like receptor 3 (TLR3) and trigger interferon responses like long, double-stranded RNA and its analogues, such as poly(LC)1 . Recently, the safety of siRNAs has been questioned because they have been shown to • y c trigger an interferon response in cultured cells ' . We show here that it is possible to administer naked, synthetic siRNAs to mice and down-regulate an endogenous or exogenous target while not inducing an interferon response. Synthetic siRNAs that are 21-23 nucleotides (nt) in length have been shown to effectively silence specific target genes by promoting mRNA degradation in cultured mammalian cells and mice6 '9. In cultured cells, there are reports of non- 161 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. specific gene target effects that include off-target gene suppression and up-regulation of type I interferons (IFNs) a and p2 " 5 ,1 0 . Hypotheses for the IFN response include recognition of the siRNA by Toll-like receptors 3 (TLR3) and 9 (TLR9) and/or induction of protein kinase PKR pathways. Toll-like receptors play critical roles in the detection of microbial infection in • 1119 » mammals by recognizing conserved microbial structures ' . Both single- (ssRNA) and double-stranded RNA (dsRNA) are molecular structures associated with viral infection; while ssRNA has been shown to interact with TLR71 3 "1 5 and TLR81 5 , dsRNA is the ligand for TLR3. Polyinosinic acid:polycytidylic acid (poly(I:C)), an analogue of dsRNA, is recognized by TLR31 ,1 6 ,1 7 and elicits immune responses in mice1 ,1 6 (TLR3 does not recognize ssRNA or double-stranded DNA (dsDNA)). Since siRNA duplexes are dsRNA, they have the potential to elicit immune response patterns different from all previously used nucleic acid therapeutics. TLR9 can recognize 2’-deoxyribo(cytidine-phosphate-guanoside) (CpG) dinucleotides flanked by specific sequences in bacterial DNA and in synthetic oligonucleotides1 8 . However, synthetic oligonucleotides with dC or dG substituted by ribonucleotides have much less immune stimulation in mice1 9 . It is possible that siRNA duplexes will not be good ligands for TLR9, but this postulate remains unproven. Thus, if siRNAs are ever to become human therapeutics, it is important to establish in vivo whether siRNA duplexes produce immune responses. The intracellular signal transduction pathways of TLR3 and TLR9 are known to promote transcription of genes regulated by the NFkB transcriptional activator that 162 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 "7 O rt O 1 include cytokines such as interleukin-12 (IL-12) ” ' . We compared the effect of poly(I:C) and siRNA on cultured RAW-264.7 cells, a mouse monocyte/macrophage line that can be induced to secrete cytokines1 or express an NFidB-dependent reporter gene1 6 by stimulation with poly(I:C). We exposed RAW-264.7 cells to various doses of either poly(I:C) or synthetic siRNA duplexes (siGL3) for 24 h and measured levels of secreted IL-12(p40) in cell supernatants by ELISA (Fig. la). LPS (lipopolysaccharide), that is known to induce a strong IL-12 response through interaction with TLR42 2, was used as a positive control for the detection of IL-12 in the assay. At all three doses examined, poly(I:C) invoked a clear IL-12 response. In contrast, synthetic siRNA failed to induce a measureable IL-12 response at any dose. Pre-treatment of poly(I:C) with RNase (Supplementary Fig. 1 online) completely abrogated the IL-12 response. The effect of siRNA on IL-12 secretion was further examined in female BALB/c mice. Administrations of synthetic siRNA duplexes or poly(I:C) were performed as intraperitoneal (IP) injections or via the tail vein using either “low- pressure” (1% v/w, “LPTV”) or “high-pressure” (10% v/w, “HPTV”) methods2 3 '2 6 and blood was collected 2 h post-injection. ELISA analyses of plasma IL-12 levels show, for each of three different injection types, a strong induction by poly(I:C) that is absent with either siRNA (siFAS or siGL3) or RNase-treated poly(I:C), even when the siFAS dose is increased up to three-fold (Fig. lb and lc). Further, injected synthetic siRNAs (siFAS and siCMYC) fail to induce any significant changes in 163 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. critical blood chemistries or liver enzyme levels except for the cases where the HPTV method of injection is used (Supplementary Fig. 2 online). The siFAS sequence does not contain any CpG motifs while the siGL3 and siCMYC sequences contain two and three, respectively. No IL-12 response is observed here as the number of CpG repeats is increased from zero to three It is known that TLR3 is located both on the cell-surface and intracellularly in human fibroblasts but remains intracellular in others such as monocyte-derived 9 7 immature dendritic cells . CpG-containing oligonucleotide/TLR9 interactions require internalization of the nucleic acid within TLR9-bearing cells28. Thus, proof of nucleic acid stability and intracellular localization in the mice are necessary. In vitro stability studies show that -50% of siRNA is still intact after exposure to serum for 2 h (Supplementary Fig. 3 online) and down-regulation of genes can occur for several days in cultured cells (see for example ref. 29 and specifically for the RAW- 264.7 cells the data are provided in Supplementary Fig. 4 online). Additionally, we injected BALB/c mice with a luciferase-containing plasmid (encoding the firefly luciferase gene under the control of the liver-specific hAAT promoter with ApoE HCR enhancer) alone or co-injected this plasmid with siRNA that contains the appropriate sequence to perform luciferase down-regulation (siGL3) or an unrelated sequence (siCNTL); all injections were HPTV. The in vivo luciferase expression was • • • 29 31 followed for two weeks post-injection by live whole-animal imaging ' . A representative image containing one mouse from each of the three treatment groups is shown in Fig. 2a. The cumulative signal from each mouse was quantified at each 164 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. time-point and the results are plotted in Fig. 2b (see also Supplementary Fig. 5 online). Mice co-injected with the luciferase-targeting siRNA display a significant (P <0.1 or better for all timepoints) down-regulation of luciferase expression that is not observed with the control siRNA. The sequence-specific RNAi demonstrates that intact siRNA duplexes do reach intracellular targets and are functional at the conditions of our studies. We also show in Fig. 2c that HPTV injection of siFAS reduces the level of Fas mRNA in mouse liver. This down-regulation was not seen upon LPTV injection of the same siRNA at the same dose, consistent with previous observations that high-pressure is required to achieve down-regulation of a target gene by siRNA in mice, even if the siRNA is chemically stabilized . The HPTV method of administrating siRNA provides for cellular uptake and gene down-regulation in livers of mice ' . In addition to determining IL-12 concentrations, these experiments allow us to test for interferon responses. The plasma levels of IFN-a in mice exposed to siRNA (siFAS, siGL3, or siCMYC) or poly(I:C) were measured by ELISA (Fig. 3). As observed with IL-12, each siRNA failed to elicit an IFN-a response while poly(I:C) induced a strong response that was eliminated by pre-treatment with RNase. Given the stability and efficacy of multiple siRNAs, the lack of IL-12 and IFN-a expression obtained in mice suggests that the synthetic siRNAs employed in this study do not elicit an observable immune response when administered naked by LPTV, HPTV or IP methods in the amounts used here. There is the possibility that the PKR response in B ALB/c mice may be compromised relative to other mouse strains.3 4 Whitmore et al. show IL-12 responses 165 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. to poly (I:C) in vitro with RAW-264.7 cells and in serum after IP injection in C57BL/6 mice like those shown here with the same cell line and BALB/c mice.3 5 We reproduced some of our experiments performed with the BALB/c mice in C57BL/6 mice, and the results show that the effects observed are consistent within those two strains (Supplementary Fig. 6 online) Sledz et al. have shown from in vitro studies that there is an upregulation of interferon-stimulating genes upon transfection of cells with certain types of siRNAs using lipid vectors.2 Bridge et al. also measured an interferon response from expressed siRNAs in cultured cells, again using lipid delivery systems. Kim et al. provide data to show that the method of siRNA preparation can determine whether or an interferon response is observed or not in culture and that purely synthetic siRNAs C 1 / like those used here do not elicit such a response. Kariko et al. and Sioud and •5'7 Sorensen have shown that lipid delivery of synthetic siRNAs can induce immune activation in vitro and in vivo, respectively by measuring protein levels in cell supernatants and serum, respectively. Additionally, Sioud and Sorensen report from in vitro studies that only certain siRNA sequences and cell types induce the activation of TNF-a and IL-6.3 7 With naked plasmid DNA that expresses human factor IX, Hodges et al. observed that neutralizing antibodies to the human factor IX were not elicited from the use of a HPTV injection in BALB/c mice while antibodies were observed from injection in skeletal muscle.3 8 These studies suggest that there are at least four issues that need further investigation: (i) the method of siRNA preparation and its sequence, (ii) the effects of cell type, e.g., hepatocytes vs. 166 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. macrophage, (iii) the effects of intracellular trafficking between lipid (and polycation) delivered siRNAs and the HPTV method (HPTV methods may not exploit the same pathways of cellular internalization2 6 as lipids and polycations), and (iv) the effects of concentration in points (i-iii) since concentration effects have been shown to be important in in vitro studies using lipid delivery systems. What is important from the results presented here is that there is a mechanistic pathway that allows for siRNAs to down-regulate both endogenous and exogenous targets in the liver without stimulating an interferon response. Further investigation into the details of this pathway should provide insights to the development of methods to perform this type of delivery in a manner that would be relevant for human therapeutics. In conclusion, we examined the response of mice to naked siRNAs. Serum IL-12 and IFN-a levels that were observed in mice by injection of poly(I:C) (used as a positive control) were not obtained with siRNA. Pre-digestion of poly(I:C) with RNase abrogated these responses. Co-injection of siRNA and a luciferase-expressing plasmid into mice followed by in vivo whole body imaging confirms the uptake and sequence-specific function of injected synthetic siRNA. High-pressure tail vein injection of siRNA alone was also shown to down-regulate an endogenous target gene (Fas) in mice. In general, synthetic siRNAs are well-tolerated in mice. 167 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. METHODS Nucleic acids. Poly(I:C) was purchased from Amersham Biosciences. SiRNA duplexes against luciferase (“siGL3”), Fas (“siFAS”), c-myc (“siCMYC”), and a non-targeting control duplex (“siCNTL”) were purchased from Dharmacon. All came purified and pre-annealed by the manufacturer (“Option C”). Sequences (CpG motifs in bold): s iGL3 5'-CUUACGCUGAGUACUUCGAdTdT-3' 3'-dTdTGAAUGCGACUCAUGAAGCU-5' siFAS: 5'-GUGCAAGUGCAAACCAGACdTdT-3' 3'-dTdTCACGUUCACGUUUGGUCUG-5' siCMYC: 5'-UCCCGCGACGAUGCCCCUCdTdT-3' 3'-dTdTAGGGCGCUGCUACGGGGAG-5' s i CNTL: 5'-GACGUAAACGGCCACAAGUUC-3' 3'-CGCUGCAUUUGCCGGUGUUCA-5' The luciferase-encoding plasmid (containing the firefly luciferase gene under the control of the liver-specific hAAT promoter with ApoE HCR enhancer) was a generous gift of A McCaffrey and M. Kay. Where indicated, nucleic acids were pre- incubated with 50 pg/mL RNase (Roche) at 37 °C for 30 min prior to use. RAW-264.7 Studies. RAW-264.7 cells were purchased from the American Type Culture Collection and cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (heat-inactivated) and antibiotics. 5e6 cells were plated per well of a 24-well plate in medium containing 1.2% DMSO. After 24 h, mIFN-y (Sigma) was added for 8 h to stimulate TLR3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. expression2 9. Lipopolysaccharide (LPS, Sigma, 1 ng/well), poly(I:C) or siRNA (siGL3) in indicated doses was added directly to appropriate wells for 24 h prior to harvesting the supernatant. 1L-I2(p40) levels were measured by ELISA (BD Biosciences) according to the manufacturer’s instructions. Mouse Studies. Female BALB/c mice (Jackson Laboratories) were 6-8 weeks of age at the time of injection. siRNA was formulated in D5W (5% glucose in water) such that a 10% v/w injection provided a 2.5 mg/kg dose, unless otherwise indicated. Mouse tails were warmed with a heating pad prior to injection of a 0.2-mL (for “low-pressure”) or a 2-mL (for “high-pressure”) volume (per 20-g mouse) over ~3-5 sec. For co-injections of siRNA with plasmid DNA, 0.25 mg/kg DNA was delivered with 2.5 mg/kg of the appropriate siRNA. To determine luciferase levels, 0.2 mL of a 15 mg/mL solution of D-luciferin (Xenogen; in PBS) was injected intraperitoneally 10 min prior to imaging. To measure plasma cytokine levels, blood was harvested from mice 2 h post injection by cardiac puncture and plasma was isolated using Microtainer tubes (Becton Dickinson). [In preliminary experiments comparing the IL-12 response at 2 h, 6 h, and 24 h, post-injection, the maximum response was consistently observed at 2 h; therefore, data from the 2 h timepoint is used here.] IL-12(p40) and IFN-a levels (PBL Biomedical Laboratories) were measured by ELISA according to the manufacturer’s instructions. To measure Fas mRNA levels, total RNA was isolated from ~100 mg of liver tissue using the FastRNA Pro 169 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Green Kit (Qbiogene) and FastPrep Instrument (Qbiogene) for 40 sec at a speed setting of 6.0. 1 ug total RNA was first digested with amplification grade DNase I (Invitrogen) and then reverse transcribed into double-stranded cDNA using oligo-dT and Superscript II (Invitrogen) following the manufacturer’s instructions. PCR primers were designed with MacVector 7.0 (Accelrys). The primer sequences are: FAS (sense): 5'-GCAAACCAGACTTCTACTGCGATTC-3' FAS (antisense): 5-CCTTTTCCAGCACTTTCTTTTCCG-3' For each PCR reaction, 1/40 (1 pL) of the cDNA reaction was amplified in a 25 pL reaction volume containing 0.5 pM of each primer and QuantiTect SYBR Green master mix (Qiagen). The real time quantitative PCR was performed and analyzed on SmartCycler (Cepheid). Standard curves were constructed by four serial 10-fold dilutions of cDNA starting from 1/20 (2 pL) of the cDNA reaction. Initial PCR conditions were 95 °C for 900 s; followed by 40 cycles of [95 °C for 15sec, 55 °C for 30 sec, and 72 °C for 30 sec]; then a final denaturing stage of gradual increasing temperature from 60 °C to 95 °C. The denaturing stage is used to generate the melting curve of PCR products, which correlate with the size and GC content of the PCR product. A parallel internal control (p-actin) was amplified at an annealing temperature of 60 °C. All PCR products were analyzed on a 1% 170 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. agarose gel and a single band corresponding to the desired PCR product was observed in all reactions except negative controls. The reproducibility of the quantitative measurement of each sample was evaluated by at least three PCR measurements. The expression level of target gene was normalized to internal p- actin and the mean and standard deviation of the target/p-actin ratios were calculated for sample-to-sample comparison. 171 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS The authors would like to thank Jean Lee and Hu Wong of the Clinical Chemistry Laboratory of the Department of Pathology, Laboratory Medicine at Children’s Hospital-Los Angeles for CBC and liver panel analyses. We thank Anton McCaffrey and Mark Kay at the Stanford Medical School for a donation of the plasmid used in our studies. J.D.H. acknowledges the Whitaker Foundation for a doctoral fellowship. S.H. is supported by an endowment in Molecular Pathology from the Las Madrinas Foundation at CHLA. 172 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. REFERENCES: 1. Alexopoulou, L., Holt, A.C., Medzhitov, R. & Flavell, R.A. Recognition of double-stranded RNA and activation of N F -kB by Toll-like receptor 3. Nature 413, 732-738 (2001). 2. Sledz, C.A., Holko, M., de Veer, M.J., Silverman, R.H. and Williams, B.R.G. Activation of the interferon system by short-interfering RNAs. Nat. Cell Biol. 5:771-772 (2003). 3. Bridge, A.J., Pebemard, S., Ducraux, A., Nicoulaz, A.-L. & Iggo, R. Induction of an interferon response by RNAi vectors in mammalian cells. Nat. Genet. 34:263-264 (2003). 4. Moss, E.G. and Taylor, J.M. Small-interfering RNAs in the radar of the interferon system. Nat. Cell Biol. 5:834-839 (2003). 5. Kim, D.-H. et al. Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase. Nat. Biotechnol. 22:321-325 (2004). 6. Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494-498(2001). 7. Novina, C.D. et al. siRNA-directed inhibition of HIV-1 infection. Nat. Med. 8:681-686 (2002). 8. Wilda, M., Fuchs, U., Wossmann, W. and Borkhardt, A. Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi). Oncogene 21:5716-5724 (2002). 9. Vickers, T.A. et al. Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. J. Biol. Chem. 278:7108-7118 (2003). 10. Jackson, A.L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21:635-637 (2003). 11. Medzhitov, R. Toll-like receptors and innate immunity. Nat. Rev. 1:135-145 (2001). 12. Zuany-Amorim, C., Hastewell, J. and Walker, C. Toll-like receptors as potential therapeutic targets for multiple diseases. Nat. Rev. 1:797-807 (2002). 13. Lund, J.M. et al. Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc. Natl. Acad. Sci. USA 101:5598-5603 (2004). 14. Diebold, S.S. et al. Innate Antiviral Responses by Means of TLR7-Mediated Recognition of Single-Stranded RNA. Science 303:1529-1531 (2004). 15. Heil, F. et al. Species-Specific Recognition of Single-Stranded RNA via Toll like Receptor 7 and 8. Science 303:1526-1529 (2004). 16. Matsumoto, M., Kikkawa, S., Kohase, M., Miyake, K., and Seya, T. Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks double-stranded RNA-mediated signaling. Biochem. Biophys. Res. Commun. 293:1364-1369 (2002). 17. Jiang, Z. et al. Poly(dI:dC)-induced Toll-like receptor 3 (TLR3)-mediated activation of NFkB and MAP kinase is through an interleukin-1 receptor- associated kinase (IRAK)-independent pathway employing the signaling 173 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. components TLR3-TRAF6-TAK1-TAB2-PKR. J. Biol Chem. 278:16713-16719 (2003). 18. Hemmi, H. et al. A Toll-like receptor recognizes bacterial DNA. Nature 408:740-745 (2000). 19. Zhao, Q., Temsamani, J., Iadarola, P.L., Jiang, Z. and Agrawal, S. Effect of different chemically modified oligodeoxynucleotides on immune stimuation. Biochem. Pharmacol. 51:173-182 (1996). 20. Liu, L., Zhou, X., Shi, J., Xie, X. & Yuan, Z. Toll-like receptor-9 induced by physical trauma mediates release of cytokines following exposure to CpG motif in mouse skin. Immunology 110:341-347 (2003). 21. Zou, W., Amcheslavsky, A., and Bar-Shavit, Z. CpG oligodeoxynucleotides modulate the osteoclastogenic activity of osteoblasts via Toll-like receptor 9. J. Biol. Chem. 278:16732-16740 (2003). 22. Chow, J.C., Young, D.W., Golenbock, D.T., Christ, W.J., and Gusovsky, F. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J. Biol. Chem. 274:10689-10692(1999). 23. Zhang, G., Budker, V., and Wolff, J.A. High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Hum. Gene Ther. 10:1735-1737(1999). 24. Liu, F., Song, Y.K. and Liu, D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther. 6:1258-1266 (1999). 25. Lecocq, M. et al. Uptake by mouse liver and intracellular fate of plasmid DNA after a rapid tail vein injection of a small or a large volume. J. Gene Med. 5:142- 156 (2003). 26. Andrianaivo, F. et al. Hydrodynamics-based transfection of the liver: entrance into hepatocytes of DNA that causes expression takes place very early after injection. J. Gene Med. 6:877-883 (2004). 27. Matsumoto, M. et al. Subcellular localization of Toll-like receptor 3 in human dendritic cells. J. Immunol. 171:3154-3162(2003). 28. Ahmed-Nejad, P. et al. Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. Eur. J. Immunol. 32:1958- 1968 (2002). 29. Layzer, J.M. et al. In vivo activity of nuclease-resistant siRNAs. RNA 10:766- 771 (2004). 30. Wu, J.C., Sundaresan, G., Iyer, M., and Gambhir, S.S. Noninvasive optical imaging of firefly luciferase reporter gene expression in skeletal muscles of living mice. Mol. Ther. 4:297-306(2001). 31. Lewis, D.L., Hagstrom, J.E., Loomis, A.G., Wolff, J.A. and Herweijer, H. Efficient delivery of siRNA for inhibition of gene expression in postnatal mice. Nat. Genet. 32:107-108 (2002). 32. Zender, L. et al. Caspase 8 small interfering RNA prevent acute liver failure in mice. Proc. Natl. Acad. Sci. USA 100:7797-7802 (2003). 174 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 33. Song, E. et al. RNA interference targeting Fas protects mice from fulminant hepatitis. Nat. Med. 9:347-351 (2003). 34. Durbin, R.K., Mertz, S.E., Koromilas, A.E. and Durbin, J.E. PKR Protection Against Intranasal Vesicular Stomatitis Virus Infection Is Mouse Strain Dependent. Viral Immunol. 15:41-51 (2002). 35. Whitmore M.M. et al. Syntergistic Activation of Innate Immunity by Double- Stranded RNA and CpG DNA Promotes Enhanced Antitumor Activity. Cancer Res. 64:5850-5859 (2004). 36. Kariko, K., Bhuyan, P., Capodici, J. and Weissman, D. Small Interfering RNAs Mediate Sequence-Independent Gene Suppresion and Induce Immune Activation by Signaling through Toll-Like Receptor 3. J Immunol. 172:6545-6549 (2004). 37. Sioud, M. and Sorensen, D.R. Cationic liposome-mediated delivery of siRNAs in adult mice. Biochem. Biophys. Res. Commun. 312:1220-1225 (2003). 38. Hodges, B.L. et al. Long-term Transgene Expression from Plasmid DNA Gene Therapy Vectors Is Negatively Affected by CpG Dinucleotides. Mol. Ther. 10:269-278 (2004). 175 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. FIGURE LEGENDS Figure 1 IL-12 induction in RAW-264.7 cells and in mice, (a) Poly(I:C), but not synthetic siRNA, induces IL-12 secretion by RAW-264.7 cells. IL-12(p40) levels in cell culture supernatants were measured upon 24 h exposure to the indicated nucleic acids and doses. The average of three replicate treatments is presented and error bars represent one standard deviation, (b) Plasma IL-12 levels in female BALB/c mice. 2.5 mg/kg (unless otherwise indicated) of nucleic acid was injected by high-pressure (10% v/w) and plasma was collected 2 h post-injection. The average of three replicate mice is presented and error bars represent one standard deviation, (c) Effect of various types of administration. Mice received 2.5 mg/kg nucleic acid either intraperitoneally (IP) or through the tail vein via low-pressure (1% v/w, LPTV) or high-pressure (10% v/w, HPTV). The average of three replicate mice is presented and error bars represent one standard deviation. [** denotes P < 0.005, * denotes P < 0.025, # denotes P < 0.30] Figure 2 Efficacy of synthetic siRNA in mice, (a) In vivo whole-body imaging of female BALB/c mice injected by HPTV with luciferase-encoding plasmid alone (left) or with siRNA targeting luciferase (siGL3, center) or non-targeting control siRNA (siCNTL, right). These mice were imaged 2 d post-injection and 10 min after IP administration of D-luciferin (for visualization). Scale bar has units of [photons/sec], (b) Synthetic siRNA accomplishes sequence-specific RNAi of an exogenous target in mice. Mice were imaged over two weeks post-injection and total 176 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. luciferase signals were integrated, quantified, and normalized to mice that received pDNA only at each timepoint. The average of three or more replicate mice is presented and error bars represent one standard deviation. [* denotes P < 0.005 at t=2 d, # denotes P < 0.025 at t=4 d, @ denotes P < 0.05 at t=6,9 d, A denotes P < 0.1 at t= 13 d. The “pDNA + siCNTL” group was not statistically different from mice that received pDNA only at these levels of significance for any of the timepoints examined.] (c) Synthetic siRNA accomplishes sequence-specific RNAi of an endogenous target in mice. Mice were injected with siFAS by HPTV or LPTV and sacrificed 24 h post-injection. Fas mRNA levels were quantified and normalized to p-actin by RT-PCR. The average of three replicate mice is presented and error bars represent one standard deviation. [* denotes P < 0.1] Figure 3 IFN-a induction in mice. 2.5 mg/kg of indicated nucleic acids were injected either intraperitoneally (IP) or through the tail vein via low-pressure (1% v/w, LPTV) or high-pressure (10% v/w, HPTV). Plasma was collected 2 h post-injection and IFN-a levels were determined by ELISA. The average of three replicate mice is presented and error bars represent one standard deviation. [* denotes P < 0.005, # denotes P < 0.025 vs. all other groups of same injection type] Supplementary Figure 1 RNase degradation of poly(I:C). 1 pg poly(I:C) was incubated with RNase (at indicated concentrations from 0-50 pg/mL) at 37 °C for 30 min, then electrophoresed on a 1% agarose gel. 177 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Supplementary Figure 2 Synthetic siRNA does not alter mouse CBC or liver enzyme levels, (a) Alanine aminotransferase (ALT), (b) Alkaline phosphatase (ALKP). (c) Platelets (PLTs). (d) White blood cells (WBCs). For all panels, mice were injected with 5% dextrose in water (D5W) or 2.5 mg/kg indicated nucleic acids (in D5W) either intraperitoneally (IP) or through the tail vein via low-pressure (1% v/w, LPTV) or high-pressure (10% v/w, HPTV). Blood was collected 2 h post injection. Whole blood was used to determine PLT and WBC levels, while plasma was isolated and used to determine ALT and ALKP levels. The average of three replicate mice is presented and error bars represent one standard deviation. Supplementary Figure 3 Degradation kinetics of synthetic siRNA in mouse serum. 40 pmol siCNTL was incubated in 20 pL mouse serum (active) at 37 °C for indicated times before being electrophoresed on a 15% TBE gel. Supplementary Figure 4 Sequence-Specific Target Down-Regulation by siRNA in RAW-264.7 Cells. Co-lipofection of siRNA with pGL3CV in RAW-264.7 cells achieves sequence-specific luciferase down-regulation. Cultured RAW-264.7 cells were exposed to lipoplexes (with LipofectAMINE (Invitrogen), according to the manufacturer’s instructions) containing 1 pg DNA and 20 nM siRNA (where indicated) for 4 h and then lysed at 48 h for measurement of luciferase and total protein levels. The average of three replicate treatments is presented as the ratio of luciferase level (RLU; relative light units) to total protein level (mg protein) for each sample. Data are normalized to the average value for Lipo/pGL3CV samples (no 178 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. siRNA); this value (“100%”) is equal to 7.5 le6 RLU/(mg protein). Error bars represent one standard deviation. [* denotes P < 0.05 vs. all other treatment groups] Supplementary Figure 5 Synthetic siRNA accomplishes sequence-specific RNAi of an exogenous target in mice. Mice were imaged over two weeks post-injection and total luciferase signals were integrated and quantified. The average of three or more replicate mice is presented and error bars represent one standard deviation. [This is the raw data that is presented in Figure 2b, presented here without further normalization.] Supplementary Figure 6 Lack of IL-12 and IFN-a Induction by siRNA in C57BL/6 Mice Plasma IL-12(p40) and IFN-a levels in female C57BL/6 mice. 2.5 mg/kg of nucleic acid was injected by high-pressure (10% v/w) and plasma was collected 2 h post-injection. The average of three replicate mice is presented and error bars represent one standard deviation. [* denotes P < 0.005 vs. all other treatment groups] 179 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. FIGURES Figure la 900 - 800 -- 700 - 150T ~ 100 -- 50-- •V Figure lb 6000 n 5000 - 4000 - 3000 - 1000 - Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure lc 0LP7V * » poly (I: C) siCMYC D5W siFAS uninjected Figure 2a Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2b 160 140 pDNA + siGL3 pDNA + siCNTL □ 2d □ 4 d ■ 6 d ■ 9 d ■ 13 d Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2c 1.400 1.200 < I 1.000 E f 0.800 i | 0.600 a E < 8 0.400 u. 0.200 0.000 uninjected siFAS, LPTV Treatment * siFAS, HPTV Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3 x O N & * > ' & o ' □ HPTV I LPTV IIP j S _ 184 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. SUPPLEMENTARY FIGURE I: RNase DIGESTION OF POLY(I:C) and SIRNA 0 [RNase] Qig/mL) 5 Q Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. SUPPLEMENTARY FIGURE 2: CBC AND LIVER PANEL RESULTS 100000 I ! L P T V 10000 H P T V 250 -| 200 - J 5 150 - ST * -j 100 - £ 50 - 0 - I □ L P T V H P T V D5W poly(l:C) siFAS siCMYC uninjected 186 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 700 - C L 200 D5W poly(l:C) siFAS LPTV H P T V uninjected □ IP h l p t v I HPTV poly(l:C) siFAS uninjected Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. SUPPLEMENTARY FIGURE 3: siRNA SERUM STABILITY 0 l h 2 h 4 ti B h • . . • . . . * ■AAiiAs-.'T ■ '■ ■ ■ ■ ■ s*5*c '<£& v "^5 ' --misixnsn mxjxsmirm* ' -- SUPPLEMENTARY FIGURE 4: SEQUENCE-SPECIFIC TARGET DOWN- REGULATION BY siRNA IN RAW-264.7 CELLS Luciferase Levels in RAW-264.7 Cells O 180 S 1 6 0 - f 140 - Q. Tre atm e n t [48 h] 188 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. SUPPLEMENTARY FIGURE 5: QUANTITATION OF LUCIFERASE DOWN-REGULATION IN VIVO Luciferase Expression 1.00E+11 1.00E+10 - 0 w 1 1.00E+09 o 3 1.00E+08 1.00E+07 B pDNA only ■ pDNA + siGL3 □ pDNA + siCNTL TimS (d) 9 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. SUPPLEMENTARY FIGURE 6: LACK OF IL-12 AND IFN-a INDUCTION BY siRNA IN C57BL/6 MICE PL-12] in C57BL/6 Plasma 1000 n ^ 900 O ) 3 800 ^ 700 S T 500 Hi 1 I D5W, HFTV siG L3, HFTV Treatment [2 h] poly(l:C), HFTV PFN-a] in C57BL/6 Plasma 800.00 E 600.00 « | 400.00 -| O l 200.00 0.00 D6W, HFTV siGL3, HFTV Treatment [2 h] * poly(l:C), HFTV 190 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Appendix 3. All primers used in this study Targets Forward Primers Reverse Primers MSX1 5 ’ -TGCTCC AGTTTC ACCTCTTTGC-3 ’ 5’-AACCTCTCTGCCCTCAGTTTCC-3’ EWS-FLI1 5 ’ -CGACT AGTT ATGATC AGAGC AGT-3 ’ 5 ’ -CCGTTGCTCTGT ATTCTT ACTG A-3 ’ CoREST 5'-ATGGCAACAGCAGCAGCAACTC-3' 5'-GGCAATGGCAATGTATTCATCC-3' NPR 5'-TGCTGGGTCAAGTGTCTCATCATAC-3' 5'-CAAGTGCTGTCACCTCCTTCCTAAG-3' JAK1 5'-CAGGTCTCCCACAAACACATCG-3' 5'-ACC AGGTCTTT ATCCTCC AAGT AGC-3' CITED2 5'-TCTGTCTTGGCTrTGGCGTTC-3' 5'-ATTAGGGCGTTGAAGGCGTG-3’ MAPT 5'-TGTGGCTCATTAGGCAACATCC-3' 5, tctgtcttggctttggcgttc-3' CYCLIN D1 5 ’ -CGC ACGATTTC ATTGAAC ACTT-3 ’ 5 ’ -CGGATTGG AAAT AC TTCACAT-3’ CYCLIN D2 5 ’ -TTGTCTC AAAGCTTGCC AGGA-3 ’ 5 ’ -CGACTTGGATCCGTC AC GTT-3’ CYCLIN D3 5 ’-CCTCTGTGCTACAGATTATACCTTTGC-3 ’ 5’-TTGCAC TGC AGCCCC AAT-3 ’ P-ACTIN 5 ’ -GCACCCCGTGCT GCTGAC-3’ 5 ’ -C AGTGGT ACGGCC AGAGG-3 ’ 191 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

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Asset Metadata

Creator Hu, Siwen (author)

Core Title Characterization of the EWS -FLI1 fusion protein in the tumorigenesis of Ewing's family of tumors

Contributor Digitized by ProQuest (provenance)

School Graduate School

Degree Doctor of Philosophy

Degree Program Pathology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, molecular,health sciences, oncology,health sciences, pathology,OAI-PMH Harvest

Language English

Advisor Triche, Timothy J. (committee chair), Dubeau, Louis (committee member), Epstein, Alan L. (committee member), Hinton, David (committee member), Warburton, David (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-572237

Unique identifier UC11335732

Identifier 3155425.pdf (filename),usctheses-c16-572237 (legacy record id)

Legacy Identifier 3155425.pdf

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Document Type Dissertation

Rights Hu, Siwen

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

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