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Cell and Tumor Biology |
Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, California
Requests for reprints: Timothy J. Triche, Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, Keck School of Medicine, University of Southern California, Box 43, 4650 Sunset Boulevard, Los Angeles, CA 90027. Phone: 323-669-4516; Fax: 323-667-1123; E-mail: triche{at}usc.edu.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The occurrence of EWS-FLI1 (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 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]; ref. 3) and they can be induced to undergo neural differentiation by various differentiating agents (4, 5).
EWS-FLI1 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 (6). However, it was later discovered that simple transfection of the EWS-FLI1 gene was generally lethal and certainly did not accelerate cell proliferation (7, 8). 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 EWS-FLI1–mediated transformation.
In contrast to its role in oncogenesis, EWS-FLI1 seems to inhibit tissue-specific differentiation. Forced EWS-FLI1 expression inhibited osteogenic and adipogenic differentiation in marrow stromal cells (9), myogenic differentiation in C2C12 cells (10), and sympathetic neural differentiation in neuroblastoma cells (11). Interestingly, tumors formed by EWS-FLI1–transformed NIH3T3 cells, an immortalized murine fibroblast line, acquired a certain degree of neural features and a small round cell morphology, which is typical of EFT but distinct from fibrosarcomas (12). This suggests a possible role of EWS-FLI1 in inhibiting tissue-associated differentiation but promoting an Ewing-specific neurectodermal differentiation program in these tumors. Further evidence is a group of biphenotypic soft tissue sarcomas. They contain the same EWS-FLI1 or EWS-ERG fusions and manifest a lesser degree of myogenic differentiation than rhabdomyosarcoma with no translocations whereas displaying some neural features (13–15).
In this study, we established a tetracycline-regulated EWS-FLI1 expression model in RD, an embryonal rhabdomyosarcoma cell line with marked myogenic differentiation, to test the hypothesis that EWS-FLI1 fusion protein is responsible for the observed primitive neuroectodermal phenotype of EFT, by the regulation of genes involved in cell proliferation and differentiation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tumors and cell lines. Frozen primary tumor tissues were obtained before therapy from Children's Hospital Los Angeles (CHLA). All cell lines were obtained from our cell bank and cultured in RPMI with 10% fetal bovine serum (Invitrogen). Fusion gene status was tested by RT-PCR. Among the 16 EFT, 36 rhabdomyosarcoma, 10 neuroblastoma, and 20 osteosarcoma samples used for gene expression analysis, EFT samples express either EWS-FLI1 or EWS-ERG, whereas the alveolar rhabdomyosarcoma samples express either PAX3-FKHR or PAX7-FKHR. TC32 and TC71 are EFT lines with type I EWS-FLI1 fusion. RD is an embryonal rhabdomyosarcoma line. CHP126 is a neuroblastoma line.
Microarray analysis. For tumor samples, tissues that had >90% tumor cells were chosen. For cultured cell lines, RNA was harvested when cell confluence was 50% to 60%. Total RNA were extracted (RNA STAT-60, Tel-Test), 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 manual at CHLA microarray core facility. The Affymetrix HU95av2 arrays contain probes for 
12,600 human full-length genes and ESTs. Chip performance, background levels and presence/absence calls were first assessed using Microarray Suite software (Affymetrix, Santa Clara, CA). Cell files were normalized with ProbeProfiler software (Corimbia, Berkeley, CA) as described before (16). Bioinformatical analyses were done with Genetrix analysis software (Epicenter Software, Pasadena, CA).
Real-time quantitative reverse transcription-PCR. Total cellular RNA was isolated using RNA STAT-60 (Tel-Test) when cells reach 50% to 60% confluence. cDNA was synthesized from 2 µg of DNase I (Invitrogen)–treated total RNA in a 42-µL reaction volume using oligo-dT and Superscript II (Invitrogen) for 60 minutes at 42°C following suppliers' instructions. PCR primers were designed with MacVector 7.0 (Accelrys, San Diego, CA). The sequences are:
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Antibodies and Western blot analysis. Cells were lysed in immunoprecipitation buffer and centrifuged for 15 minutes at 14,000 x g. Solubilized proteins in the supernatant were quantified using bicinchoninic acid protein assay (Bio-Rad, Richmond, CA). Sixty micrograms of total cellular protein were loaded per lane, separated by precast SDS-PAGE (Invitrogen), and transferred to a polyvinylidene difluoride 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 horseradish peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Bound secondary antibodies were detected using an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ). Monoclonal antibodies against FLI1, MyoD, and Myogenin were from BD PharMingen (San Diego, CA) and MAPT from Neomarkers (Fremont, CA). Polyclonal ß-actin antibody was from Santa Cruz Biotechnology.
Xenograft experiments. Four- to 6-week-old severe combined immunodeficient mice were injected s.c. 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 quantitative reverse transcription-PCR (QRT-PCR).
Electron microscopy. Electron microscopy (EM) was carried out by the standard procedure at the CHLA EM laboratory. 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 epon (Embed-812, Electron Microscopy Sciences, Hatfield, PA). One-micrometer-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 ultrastructural examination. Ultrathin 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 µm) and deparaffinized. Sections were boiled 15 minutes in H2O for antigen retrieval, quenched with 3% hydrogen peroxide for 5 minutes. A M.O.M. Immunodetection Kit (Vector Laboratories, Burlingame, CA) was used for detecting monoclonal mouse anti-human antibodies (MyoD1, DAKO, Carpinteria, CA; Myogenin, DAKO; Muscle Actin, Cell Marque, Hot Springs, AR; Desmin, DAKO; MIC2, DAKO; and TAU, Neomarkers) following 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 followed by 30 minutes incubation with biotinylated anti-mouse or anti-rabbit IgG secondary antibody. Sections were exposed to diaminobenzidine peroxidase substrate (Sigma, St. Louis, MO) 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.
RNA interference. Small interfering RNA (siRNA) against EWS-FLI1 breakpoint (siEFBP2) and a nontargeting control siRNA (C8) were obtained from Dharmacon Research (Lafayette, CO). Sequence of siEFBP2 was reported before (18). siEFBP2 or C8, complexed with TransIT-TKO (Mirus) in Opti-MEM I (Invitrogen) following manufacturer's directions, were applied to RD-EF cells or TC32 cells 30% confluent in RPMI with 10% fetal bovine serum but without antibiotics in 6-well plates, to give a final concentration of 100 nmol/L. For RD-EF cells, 12 hours after transfection, the cells were induced by tetracycline or ethanol for another 24 hours before RNA was harvested. For TC32 cells, the same transfection was repeated after 24 hours and RNA was harvested after another 24 hours. Gene expression was assessed by QRT-PCR.
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Results |
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EWS-FLI1 expression in RD cells revealed a shift in global gene expression pattern with induction of EFT markers. To provide visual representation of the samples based on gene expression, we used principal component analysis (PCA; ref. 19) 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. Continued expression of EWS-FLI1 chimeric gene induced a progressive shift away from both RD cells and ethanol-treated controls (Fig. 1C). All of the controls cluster together without an apparent pattern. However, tetracycline-treated samples cluster in a distinct pattern that correlates directly with the induced time. Longer exposure to tetracycline resulted in a greater accumulation of EWS-FLI1 protein, and data points separated progressively further from that of the controls. These data indicated a fundamental difference in gene expression pattern between the EWS-FLI1–expressing cells and the controls.
Detailed analysis of the differentially expressed genes revealed several noteworthy genes previously identified as EFT markers (CD99, ref. 20; CCK, ref. 3; and STEAP, ref. 21), or EWS-FLI1 downstream genes (c-MYC, ref. 22; ID2, refs. 23, 24; and Cyclin, ref. 25), all highly expressed in EFT, and also highly up-regulated in tetracycline-treated RD-EF cells (Figs. 1D and 2C, m-r). Interestingly, the up-regulation of c-MYC and ID2 (6 hours) preceded Cyclin D1 (18 hours), which implied that these two genes are early response genes and Cyclin D1 is possibly a secondary target of EWS-FLI1. EWS-FLI1 down-regulates transforming growth factor ßRII (TGFßRII; ref. 26) and P21 (27). It seemed that ethanol itself could regulate the expression of TGFßRII. However, tetracycline-treated RD-EF cells showed more down-regulation than ethanol-treated cells. P21 expression decreased dramatically up to 18 hours. Thus, expression of EWS-FLI1 resulted in a marked shift in global gene expression, with induction and suppression of several known EFT markers and EWS-FLI1 target genes.
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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 EWS-FLI1. RD cells normally form bona fide rhabdomyosarcoma tumors with marked terminal rhabdomyogenesis, whereas EFT cells are largely undifferentiated with scant neural differentiation marked by clusters of cytoplasmic dense core granules. Strikingly, RD cells that express EWS-FLI1 show near complete suppression of the myogenic phenotype with concomitant neural differentiation, marked by the appearance of neuritic processes containing dense core granules (Fig. 2B). H&E staining of RD-EF xenografts showed a structureless array of biphasic cell population (small dark and large light types) with scant cytoplasm (Fig. 2C, b), which is typical of EFT tumors (Fig. 2C, c). On the other hand, RD xenografts still showed typical rhabdomyosarcoma tumor phenotype (a diffuse infiltrate of small round-to-spindled cells in a collagenous stroma, Fig. 2C, a). Clearly, the EWS-FLI1 gene has a potent neural differentiating effect at the expense of the existing tissue differentiation. This dual effect has not been previously recognized.
Appearance of an Ewing-like neuroectodermal phenotype was 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 >8-fold after EWS-FLI1 expression (Fig. 3A; P < 0.001). MAPT is expressed exclusively in the axons of neurons and promotes microtubule assembly and stability. Both EFT and neuroblastoma but not rhabdomyosarcoma express MAPT at high levels. Keratin 18 is an epithelial structural protein. The up-regulation of these two genes implied that EWS-FLI1 induced a neuroectodermal phenotype in mesodermal RD cells, consistent with the morphologic results reported above. Western blot and immunohistochemical analysis showed that the MAPT protein started to accumulate at 24 hours and reached peak level at 48 hours (Figs. 2C, j-l and 3B), which suggested that MAPT takes part in the morphologic change induced by EWS-FLI1 expression. Moreover, the up-regulation of CCK (Figs. 1D and 2C, m-o), whose expression differentiates the parasympathetic neural phenotype of EFT from the sympathetic neural phenotype of neuroblastoma, clearly indicated that a specific Ewing-like parasympathetic neural phenotype was imposed in RD cell by EWS-FLI1 expression.
We also examined the effect of EWS-FLI1 expression on myogenesis, cognizant of the profound loss of morphologic evidence of myogenesis noted in Fig. 2. 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 cholinergic receptor 
and cholinergic receptor 
. Two other myogenic transcription factors, myf5 and myf6, were unaffected by EWS-FLI1 expression (Fig. 3C). Interestingly, this expression pattern of myogenic transcription factors is reminiscent of what was seen in biphenotypic sarcomas expressing EWS-FLI1 (13). Protein level confirmation of the down-regulation of MyoD and Myogenin by Western blot is illustrated in Fig. 3D, where a nearly inverse relationship between levels of EWS-FLI1 protein and these two transcription factors was noted. Immunohistochemical staining is consistent with the microarray and Western results (Fig. 2C, d-i). Moreover, Cyclin D3, the major D-type cyclin expressed in rhabdomyosarcoma (17) and associated with muscle differentiation (28, 29), was down-regulated to an almost undetectable level after EWS-FLI1 induction, in parallel with the increase of cyclin D1 (the major D-type cyclin in EFT; Figs. 1D and 3C; Supplementary Fig. 1B, ref. 17). These data indicate that EWS-FLI1 expression induces a profound down-regulation of the muscle differentiation program.
Comparison of up-regulated genes in RD-EF system and highly expressed genes in Ewing's family tumors revealed genes crucial in neural crest development and WNT signaling pathway. In an attempt to specify genes that were significantly up-regulated by EWS-FLI1, we first identified a list of 865 "EFT signature genes" of the 12,625 probe sets on the HU95av2 arrays, that are highly associated with primary EFT from patients (P 
0.001) and are expressed at least 2-fold higher than in the rest of the tumors. PCA analysis showed that at 24 and 36 hours, tetracycline-treated RD-EF cells were completely separated from the controls and the EWS-FLI1 protein level was stable and comparable with the original EFT cell lines. Thus, we compared the gene expression pattern of the T24 and T36 samples with the controls, including untreated and all of the ethanol-treated samples, and selected 370 genes that are highly associated with T24 and T36 (P 0.001). Of these EWS-FLI1–induced genes, 109 (30%) are also signature genes of the EFT (Table 1; Fig. 4D; Supplementary Fig. 2). Hierarchical clustering of the RD-EF samples separated these 109 genes in a temporal manner (Fig. 4A). Two main gene expression patterns within the time course were identified: genes that were up-regulated at early time points (T6 and T12) and 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 EWS-FLI1–up-regulated genes are important for neural crest development, such as EGR2 (Krox20), MSX1, 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 EWS-FLI1 is to invoke a form of neurogenesis. Other neural associated genes induced by EWS-FLI1 included neuronal pentraxin receptor, synaptotagmin I, SMA5, presenilin 1, as well as GABARAPL, XPNPEP1, DPYSL2, CSRP1, and OLFM1.
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Up-regulation of neural crest genes was confirmed in another cell lineage expressing EWS-FLI1. Our result suggests that EWS-FLI1 is a potent differentiation factor that blocks a preexisting commitment to myogenesis in rhabdomyosarcoma whereas 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 EWS-FLI1. The fact that these genes are also highly expressed in primary EFT precludes the possibility that this is merely a nonspecific effect after the muscle differentiation program was inhibited. However, because cell context is very important in extrapolating EWS-FLI1's function, we have pooled our RD-EF gene expression data with comparable expression profiles derived from primary, untreated EFT, and a similar data set derived from EWS-FLI1 transfectants in a human normal foreskin fibroblast model (HNFF-EF) kindly provided by the authors (8). Using a cutoff of P < 0.001 (and expression >2-fold increased in the case of primary EFT), we have done a multiple t test analysis of each data set to identify the genes that meet these selection criteria and compared results in a Venn diagram (Fig. 5B). A final 46 genes common to all three datasets was identified (highlighted in Table 1). These genes are of especial interest, because they are clearly up-regulated by expression of EWS-FLI1, independently of cellular background, normal or malignant. Strikingly, the majority of the neural crest differentiation–related genes identified in RD-EF model were also up-regulated by EWS-FLI1 expression in this human fibroblast background. Fourteen of the 46 genes are associated with neurogenesis, and even more strikingly, of the seven genes most strongly associated with the EWS-FLI1 expression in the model systems (P < 0.000000001), the majority (5) are involved in neural differentiation, particularly neural crest development (Table 1; ref. 31). Thus, there is a strong association between neurogenesis and EWS-FLI1 expression. These data clearly indicate that neural differentiation induced by expression of EWS-FLI1 is independent of cellular background in which the gene is expressed. It excludes the possibility of a nonspecific neural up-regulation as a result of the down-regulation of myogenesis and indicates a general effect of EWS-FLI1 on differentiation.
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Discussion |
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Our study showed that EWS-FLI1 has a profound effect on cell differentiation as well as proliferation. It can induce an Ewing-like neural crest phenotype in multiple cellular contexts whereas 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." Otherwise conspicuous terminal skeletal muscle differentiation is nearly completely lost in RD cells expressing EWS-FLI1, 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, MSX1 (34), and c-MYC are all neural crest markers during development. Krox20 knockout mice have severely defective myelination of peripheral nerves (35). MSX1–/– mice have deficiencies in neural crest derivatives (36). C-MYC was shown an essential early regulator of neural crest development and knockdown of c-MYC by antisense ODN resulted in a loss of neural crest precursors in Xenopus embryos (37). Overexpression of ID2 can drive ectodermal cells into a neural crest phenotype instead of the epidermal lineage (38). 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 (39–41). Cadherin 11 and Rho family GTPases are both related to delamination of neural crest cells from the neural tube (42–44). RUNX3 is an important regulator of the axonal projections of a subpopulation of dorsal root ganglion neurons (45).
Expression of neural structural genes, such as neuronal pentraxin receptor, synaptotagmin I, and MAPT, is also highly induced. CCK is a neuropeptide and is highly expressed in EFT (46). 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 EWS-FLI1 in a neuroblastoma cellular background (11) suppresses sympathetic neural differentiation, the hallmark feature of neuroblastoma cells, whereas MIC2 (CD99), c-MYC, and keratin 18 were all markedly up-regulated, similar to what we observed in this study. These data exclude the possibility of a nonspecific neural up-regulation and indicate a general effect of EWS-FLI1 on differentiation. The high expression of all these critical neural crest associated genes provided further evidence for a parasympathetic neural crest differentiation program in EFT.
Because James Ewing described Ewing's sarcoma >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 multipotent 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 (MSC), which are classic mesodermal derivatives, have been shown multidifferentiated in addition to being multipotent and could be induced to differentiate into neurons (47). Considering that most EFT occurs in bone and soft tissue, MSC serves as one possible source of the cells susceptible of EWS-FLI1 transformation. A similar argument may be true in soft tissue, where pluripotent stem cells have also been described.
Despite several years of effort by multiple investigators, the basic mechanism whereby EWS-FLI1 expression results in the tumor entity that we recognize as EFT 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. 5B). 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-FLI1 (24, 48). It is possible that EWS-FLI1 can up-regulate both genes directly and overexpression 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 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-FLI1 (27) 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 dedifferentiate terminally differentiated murine myotubes by repressing MyoD, Myogenin, cyclin D3, p21 and up-regulating cyclin D1 with minimal effect on cell proliferation (49, 50). Similar effects seen after EWS-FLI1 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 because its up-regulation starts at 18 hours after EWS-FLI1 induction, 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 autosuppression could be counteracted by CBP/p300, which is the necessary partner of CITED2 for TFAP2 coactivation (51, 52).
The striking incidence of WNT signaling–associated genes identified in this analysis cannot be due to chance alone. WNT signaling has been found to be very important in neural crest development (53). WNTs could function as endogenous neural crest inducers in avian embryos, and inhibition of this pathway could block neural crest precursor formation in Xenopus (54). However, the classic WNT/ß-catenin mediated signaling through TCF/LEF target genes is not likely active in EFT tumors (Supplementary Fig. 2). Can EWS-FLI1 hijack the classic WNT signaling downstream of ß-catenin? Or does it activate a noncanonical WNT pathway, whose mechanism is still largely unknown? Or has a completely different, currently undescribed, pathway been used? Increased knowledge of the noncanonical WNT pathways will help to answer these questions.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Darkin Chan and Minerva Mongeotti (EM Laboratory) for EM processing; Morgan Wu (clinical pathology laboratory) for immunohistochemical staining; Betty Schaub, Xuan Chen, and Sitara Waidyaratne (Microarray Core) for microarray processing; and Dr. George Mcnamara (Imaging Core) for his expertise at microscopy.
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Footnotes |
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Received 11/ 4/04. Revised 3/ 1/05. Accepted 3/22/05.
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 K. Engstrom, H. Willen, C. Kabjorn-Gustafsson, C. Andersson, M. Olsson, M. Goransson, S. Jarnum, A. Olofsson, E. Warnhammar, and P. Aman The Myxoid/Round Cell Liposarcoma Fusion Oncogene FUS-DDIT3 and the Normal DDIT3 Induce a Liposarcoma Phenotype in Transfected Human Fibrosarcoma Cells Am. J. Pathol., May 1, 2006; 168(5): 1642 - 1653. [Abstract] [Full Text] [PDF]
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