William A. May

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.

EWS/FLI-1 is a chimeric protein formed by a tumor-specific 11;22 translocation found in both Ewings sarcoma and primitive neuroectodermal tumor of childhood. EWS/FLI-1 has been shown to be a potent transforming gene, suggesting that it plays an important role in the genesis of these human tumors. We now demonstrate that EWS/FLI-1 has the characteristics of an aberrant transcription factor. Subcellular fractionation experiments localized the EWS/FLI-1 protein to the nucleus of primitive neuroectodermal tumor cells. EWS/FLI-1 specifically bound in vitro an ets-2 consensus sequence similarly to normal FLI-1. When coupled to a GAL4 DNA-binding domain, the amino-terminal EWS/FLI-1 region was a much more potent transcriptional activator than the corresponding amino-terminal domain of FLI-1. Finally, EWS/FLI-1 efficiently transformed NIH 3T3 cells, but FLI-1 did not. These data suggest that EWS/FLI-1, functioning as a transcription factor, leads to a phenotype dramatically different from that of cells expressing FLI-1. EWS/FLI-1 could disrupt normal growth and differentiation either by more efficiently activating FLI-1 target genes or by inappropriately modulating genes normally not responsive to FLI-1.

Identification of target genes for the Ewing's sarcoma EWS/FLI fusion protein by representational difference analysis.

The EWS/FLI-1 fusion gene results from the 11;22 chromosomal translocation in Ewings sarcoma. The product of the gene is one of a growing number of structurally altered transcription factors implicated in oncogenesis. We have employed a subtractive cloning strategy of representational difference analysis in conjunction with a model transformation system to identify genes transcribed in response to EWS/FLI. We have characterized eight transcripts that are dependent on EWS/FLI for expression and two transcripts that are repressed in response to EWS/FLI. Three of the former were identified by sequence analysis as stromelysin 1, a murine homolog of cytochrome P-450 F1 and cytokeratin 15. Stromelysin 1 is induced rapidly after expression of EWS/FLI, suggesting that the stromelysin 1 gene may be a direct target gene of EWS/FLI. These results demonstrate that expression of EWS/FLI leads to significant changes in the transcription of specific genes and that these effects are at least partially distinct from those caused by expression of germ line FLI-1. The representational difference analysis technique can potentially be applied to investigate transformation pathways activated by a broad array of genes in different tumor systems.

E-cadherin cell-cell adhesion in ewing tumor cells mediates suppression of anoikis through activation of the ErbB4 tyrosine kinase.

Ability to grow under anchorage-independent conditions is one of the major hallmarks of transformed cells. Key to this is the capacity of cells to suppress anoikis, or programmed cell death induced by detachment from the extracellular matrix. To model this phenomenon in vitro, we plated Ewing tumor cells under anchorage-independent conditions by transferring them to dishes coated with agar to prevent attachment to underlying plastic. This resulted in marked up-regulation of E-cadherin and rapid formation of multicellular spheroids in suspension. Addition of calcium chelators, antibodies to E-cadherin (but not to other cadherins or beta(1)-integrin), or expression of dominant negative E-cadherin led to massive apoptosis of spheroid cultures whereas adherent cultures were unaffected. This correlated with reduced activation of the phosphatidylinositol 3-kinase-Akt pathway but not the Ras-extracellular signal-regulated kinase 1/2 cascade. Furthermore, spheroid cultures showed profound chemoresistance to multiple cytotoxic agents compared with adherent cultures, which could be reversed by alpha-E-cadherin antibodies or dominant negative E-cadherin. In a screen for potential downstream effectors of spheroid cell survival, we detected E-cadherin-dependent activation of the ErbB4 receptor tyrosine kinase but not of other ErbB family members. Reduction of ErbB4 levels by RNA interference blocked Akt activation and spheroid cell survival and restored chemosensitivity to Ewing sarcoma spheroids. Our results indicate that anchorage-independent Ewing sarcoma cells suppress anoikis through a pathway involving E-cadherin cell-cell adhesion, which leads to ErbB4 activation of the phosphatidylinositol 3-kinase-Akt pathway, and that this is associated with increased resistance of cells to cytotoxic agents.

PDGF-C is an EWS/FLI induced transforming growth factor in Ewing family tumors

The aberrant transcription factors associated with many human malignancies function by deregulation of tumorigenic pathways. However, identification of these pathways has come slowly. Virtually all cases of Ewings Sarcoma and peripheral Primitive Neuroectodermal Tumor (PNET) are associated with aberrant transcription factors which fuse amino-terminal EWS with the DNA binding moiety of an ETS transcription factor (FLI-1 in 90% of cases). Attempts to identify the downstream targets of these chimeras in the Ewing Family Tumors (EFT) on the basis of differential gene regulation have produced little association with tumor biology. As an alternative approach, we have used highly efficient retroviral systems to biologically screen cDNA derived from cells transformed by EWS/FLI-1. We have identified the recently described PDGF-C as target of EWS/ETS transcriptional deregulation. This transcriptional deregulation is specific to EWS/FLI. PDGF-C possesses substantial biologic activity in vitro and in vivo. It is expressed in EFT cell lines and in primary tumors. Within these EFT cell lines, PDGF-C expression is dependent upon EWS/FLI activity. These results suggest that PDGF-C may be a significant mediator of EWS/FLI driven oncogenesis.

The EWS/FLI1 oncogenic transcription factor deregulates GLI1.

Ewing family tumors (EFT), classically Ewings sarcoma and peripheral primitive neuroectodermal tumor, share a common class of tumor-specific fusion genes thought to be key mediators of tumor biology. Here we demonstrate that the most common Ewings fusion, EWS/FLI1, produces transcriptional upregulation of GLI1 and its direct transcriptional target PATCHED1 in a model transformation system. This deregulation of GLI1 is common to other EWS/ets chimera and depends on the functional transcriptional regulatory domains. Inhibition of GLI1 via RNAi or via overexpression of endogenous inhibitors results in a reduction of EWS/FLI1 transformation activity. Activation of GLI1 appears to occur in a Hedgehog-independent fashion as blockade of Hedgehog signaling has only a modest effect on EFT cells. We present evidence that EWS/FLI1 upregulation of cMYC may play a role in the upregulation of GLI1 in EWS/FLI1-transformed NIH3T3 cells. Finally, we demonstrate that observations made in a model transformation system translate to an Ewing cellular background. EFT cell lines express GLI1 and PATCHED and this expression is EWS/FLI1 dependent. Inhibition of GLI1 expression via RNAi results in reduced anchorage-independent growth in an EFT cell line. GLI1 appears to be a transcriptionally deregulated target of EWS/FLI1 that mediates a portion of its tumorigenic phenotype.

GLI1 Is a Central Mediator of EWS/FLI1 Signaling in Ewing Tumors

The Ewing Sarcoma Family Tumors (ESFT) consist of the classical pathologic entities of Ewing Sarcoma and peripheral Primitive Neuroectodermal Tumor. Occurring largely in the childhood through young adult years, these tumors have an unsurpassed propensity for metastasis and have no defined cell of origin. The biology of these aggressive malignancies centers around EWS/FLI1 and related EWS/ETS chimeric transcription factors, which are largely limited to this tumor class. Much progress has been made in the identification of a network of loci whose expression is modulated by EWS/FLI1 and its congeners. To date, little progress has been made in reconstructing the sequence of direct and indirect events that produce this network of modulated loci. The recent identification of GLI1 as an upregulated target of EWS/ETS transcription factors suggests a target which may be a more central mediator in the ESFT signaling network. In this paper, we further define the relationship of EWS/FLI1 expression and GLI1 upregulation in ESFT. This relationship is supported with data from primary tumor specimens. It is consistently observed across multiple ESFT cell lines and with multiple means of EWS/FLI1 inhibition. GLI1 inhibition affects tumor cell line phenotype whether shRNA or endogenous or pharmacologic inhibitors are employed. As is seen in model transformation systems, GLI1 upregulation by EWS/FLI1 appears to be independent of Hedgehog stimulation. Consistent with a more central role in ESFT pathogenesis, several known EWS/FLI1 targets appear to be targeted through GLI1. These findings further establish a central role for GLI1 in the pathogenesis of Ewing Tumors.

Dominant negative PDGF-C inhibits growth of Ewing family tumor cell lines

Nearly all cases of Ewing Family Tumors (EFT) harbor chimeric EWS/ETS transcription factors which are thought to aberrantly regulate transcriptional targets of phenotypic consequence. We have recently demonstrated that EWS/ETS proteins up-regulate platelet derived growth factor-C (PDGF-C), a novel transforming growth factor. To determine if PDGF-C signaling contributes to the malignant phenotype of EFT cell lines, we attempted to disrupt this presumed autocrine loop. AG1296, a PDGF receptor selective tyrosine kinase inhibitor, markedly inhibits anchorage-independent growth in an EFT cell line. To effect specific disruption, we have developed a dominant negative form of PDGF-C which is appropriately secreted and processed. This mutant has greatly reduced activity as a PDGF receptor agonist. When co-expressed with PDGF-C in a fibroblast transformation model, this dominant negative dramatically inhibits anchorage-independent growth. When this mutant is expressed in EFT cell lines, there is a similar reduction in anchorage-independent growth. This demonstrates that specific inhibition of PDGF-C signaling in EFT cell lines partially reverts their phenotype. These data support a significant role of PDGF-C in the biology of EFT. They also suggest that PDGF-C driven signaling may be a possible therapeutic target of more clinically relevant tyrosine kinase inhibitors.

The clonal nature of mediastinal germ cell tumors and acute myelogenous leukemia: A case report and review of the literature

The clonal identity of a mediastinal germ cell malignant tumor and acute myelogenous leukemia is described in an 11-year-old boy in whom both tumors presented simultaneously. The relationship between these two histologically distinct malignancies is discussed in relation to this patient and 34 previously reported patients.

Analysis of clear corneal incision integrity in an ex vivo model

PURPOSE: To determine the most favorable sutureless incision configuration to minimize extraocular fluid inflow after cataract surgery. SETTING: The Wilmer Eye Institute, Baltimore, Maryland, USA. METHODS: Five fresh human eyes were used in the study. Two 27‐gauge needles connected to a saline solution bag and a digital manometer were inserted through the limbus 180 degrees from each other. Intraocular pressure (IOP) was maintained at 15 to 20 mm Hg. Three incisions were performed in different quadrants of each cornea: uniplanar 1.0 mm and 3.0 mm tunnel lengths and 2‐step 3.0 mm tunnel length. India ink was applied to the incision site, and IOP fluctuation was induced by applying pressure to the limbal area of the opposite quadrant using an ophthalmodynamometer. Imaging was performed before and after pressure application. RESULTS: The linear distance of India ink inflow after pressure application was higher than the prepressure measurements in the 1.0 mm and 3.0 mm incision groups (P = .039 and P = .023, respectively). The maximum mean of inflow after pressure application was not higher than the prepressure measurement in the 2‐step incision group (P = .105). The total ink area measured before and after pressure applications in the incisions of the 3 groups was not significantly different (P = .285). CONCLUSIONS: Intraocular pressure fluctuations may promote entry of bacteria‐size particles into the eye when 1.0 mm and 3.0 mm single‐plane incisions are performed. Stepped incisions seem to be more resistant to inflow in the presence of IOP fluctuation.

EWS/FLI function varies in different cellular backgrounds

EWS/FLI and other EWS/ets chimeric transcription factors play a central role in the biology of the Ewing family tumors. As with many oncogenes, EWS/FLI biologic activity can be demonstrated in a limited range of cellular contexts. To investigate the causes of this restriction, we demonstrate that two immortalized fibroblast lines resistant to EWS/FLI transformation, Rat1 and Yal7, express stable levels of EWS/FLI protein. Despite their resistance to EWS/FLI, Rat1 and Yal7 can be transformed by the potent EWS/FLI downstream mediator PDGF-C. In contrast to NIH3T3, the EWS/FLI resistant lines show no upregulation of PDGF-C in response to EWS/FLI, demonstrating differential EWS/FLI function in different cellular backgrounds. This phenomenon of differential function can also be demonstrated for several other NIH3T3 targets of EWS/FLI. Despite the correlation between anchorage-independent growth and PDGF-C induction, PDGF-C does not fully reproduce all aspects of the EWS/FLI phenotype in NIH3T3 cells. These results further point to the importance of PDGF-C in mediating EWS/FLI in vitro transformation and suggest caution in assuming that a transcription factor will produce identical effects in different cellular backgrounds.