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Related ESUN Articles

Ewing's Sarcoma Family of Tumors (ESFT)

Small Molecules That Can Inhibit EWS-FLI1 May Represent Novel and Specific Therapy for ESFT Patients

Novel Approaches for the Molecular Targeting of Ewing's Sarcoma Family of Tumors

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Gene Profiling Studies on Sarcomas

Related Research Grants

Analysis of NR0B1 in Ewing’s sarcoma

New Approaches for EWS/ETS Detection in Ewing’s Sarcoma

EWS/FLI and its targets in Ewing’s sarcoma:
a progress report and future directions

An ESUN Article

Stephen L. Lessnick, MD, PhD
Associate Professor of Pediatrics
Adjunct Assistant Professor of Oncological Sciences,
Investigator, Center for Children at Huntsman Cancer Institute
University of Utah School of Medicine
Salt Lake City, UT 84112

Introduction

Ewing’s sarcoma is a highly aggressive and enigmatic tumor of children and young adults (Ref. 1). While this tumor most commonly arises in and around bones, it does not appear to be of bony origin. Indeed, the type of cell that transforms into Ewing’s sarcoma is not currently known. This makes it difficult to study the process of Ewing’s sarcoma formation.

An excellent review article by R. Lor Randall, MD, "Ewing's Sarcoma Family of Tumors (ESFT)" appears in ESUN, Vol. 1, N. 3, 2004.

Tumors are generally thought to arise because of mutations in normal genes. In the case of Ewing’s sarcoma, the most important mutation is called a chromosomal translocation (Refs. 2 and 3). Chromosomes are large segments of linearly-arranged DNA that contain many genes. Humans typically have 23 pairs of chromosomes. In Ewing’s sarcoma tumor cells, chromosomes 11 and 22 have traded portions of their DNA, creating two abnormal chromosomes (Refs. 2 and 3). At the exact portion where the two chromosomes have traded genetic material, two different genes, EWS (or more properly, EWSR1) and the other called FLI, have become fused together in an abnormal way (Ref. 4). This creates a mutant protein called EWS/FLI (Ref. 4 and Figure 1). Rarely, Ewing’s sarcoma tumors will have different chromosomal translocations that generate different, but related, mutant proteins. EWS/FLI (or one of the related proteins) is found in nearly every case of Ewing’s sarcoma (Ref. 5). From here on out I will refer to EWS/FLI exclusively, but recognize that the themes discussed are conserved (or presumed to be conserved) for the other related fusions as well. EWS/FLI is only found in the tumor cells of the patient, not in the normal cells. These data suggest that EWS/FLI is the critical mutation required for the development of Ewing’s sarcoma.

Figure 1. EWS/FLI schematic. Wild-type (normal) EWS is encoded on chromosome 22, and contains an "amino-terminal domain" and a series of domains in the carboxy terminus that are likely RNA binding regions. Wild-type FLI is encoded on chromosome 11, and contains a weak transcriptional activation domain (TAD) in its amino terminus, and a DNA binding domain in its carboxy terminus. The t(11;22)(q24;q12) translocation encodes the fusion EWS/FLI protein, that contains the amino terminal domain of EWS, fused in-frame, to the carboxy terminus of FLI. In this setting, the amino terminus of EWS functions as a strong transcriptional activation domain, while FLI contributes its DNA binding domain to the fusion.

The diagnosis of Ewing’s sarcoma can be problematic. When surgeons or interventional radiologists remove a small piece of the tumor, called a biopsy, the tumor sample is given to a pathologist to evaluate for diagnosis. This involves a variety of procedures, including looking at the tumor under the microscope, and staining the tumor for proteins that are usually found in Ewing’s sarcoma. Unfortunately, neither of these approaches are perfectly specific, and diagnostic dilemmas still occur (i.e., there are cases in which a diagnosis can not be made). A complementary approach is to look for the chromosomal translocation (or its products) that is usually associated with Ewing’s sarcoma. While this approach tends to be more specific, it requires that the tumor material be of high quality, and processed in a rigorous way. Unfortunately, not all tumor biopsies are of adequate quality or processed appropriately. New approaches that are more sensitive, and more forgiving, would aid in the diagnosis of Ewing’s sarcoma.

One of the projects that we have focused on, using a grant awarded by the Liddy Shriver Sarcoma Initiative, is to develop an improved method to detect the products of Ewing’s sarcoma-associated chromosomal translocations. We have devised an approach that combines a series of new technologies to detect EWS/FLI, or any of the related mutants found in Ewing’s sarcoma. We hope that this technique will be more sensitive, and will thus allow for the detection of the fusion products in samples that are of low quality, or not processed adequately for more "typical" approaches. At this point, the technology is still quite early, but we have some promising results that suggest that the approach will be feasible.

In addition to improving the diagnosis of Ewing’s sarcoma, our lab is focused on identifying new therapeutic approaches for this disease. We believe that understanding the details of how EWS/FLI causes Ewing’s sarcoma will allow us to find new ways to treat this disease. A second project that we have been pursuing, with ongoing funding from the Liddy Shriver Sarcoma Initiative, is analyzing the EWS/FLI target gene NR0B1.

EWS/FLI is thought to function as a transcription factor (Refs. 4, 6, and 7). Transcription factors bind directly to DNA, and alter the expression of nearby genes. In order to study EWS/FLI in its "normal" cellular context, we developed a system that allowed us to analyze the function of EWS/FLI in Ewing’s sarcoma cells themselves, rather than heterologous cell types (Ref. 8 and Figure 2). We used RNA-interference (RNAi) to "knock-down" (diminish) the amount of EWS/FLI in patient-derived Ewing’s sarcoma cell lines (Ref. 9). Following the RNAi treatment, we have the ability to reintroduce EWS/FLI expression using a version that is immune to the RNAi effect. We then used high-density microarrays to identify many of the RNA transcripts whose expression levels change when EWS/FLI is knocked down in these cells (Refs. 9 and 10).

Figure 2. The "knock-down/rescue" approach. Patient-derived Ewing’s sarcoma cell lines are grown in the laboratory. These normally express EWS/FLI. Treatment with RNA interference (RNAi) results in a "knock-down" of EWS/FLI mRNA and protein levels. EWS/FLI can be re-expressed using a version that is resistant to the RNAi effect. Thus, the function of EWS/FLI can be studied in the cells in which it is normally found: Ewing’s sarcoma tumor cells.

One of the genes we found whose expression was upregulated by EWS/FLI was NR0B1 (Refs. 10 and 11). Importantly, in recent work (also supported by the Liddy Shriver Sarcoma Initiative), we also found that NR0B1 was directly regulated by EWS/FLI (in other words, EWS/FLI bound directly to the NR0B1 promoter) via a GGAA-containing microsatellite in its promoter (Ref. 12). This is interesting and unique, as microsatellites are simple repetitive elements that are located throughout the human genome, and are often considered "junk" DNA with no real function. The identification of a GGAA-microsatellite as an EWS/FLI response element demonstrates an important role for these sequences in Ewing’s sarcoma development. This work was recently published in the Proceedings of the National Academy of Sciences (Ref. 12).

The normal function of the NR0B1 protein is not entirely clear. It is important for adrenal gland development and for the development and function of the hypothalamic-pituitary-adrenal-gonadal axis. Since its first identification in 1994 (Ref. 13), a series of functions have been ascribed to the protein, including direct DNA binding and transcriptional repression (Refs. 13 and 14), inhibition of other transcription factors such as the retinoic acid receptor (Ref. 13), SF-115, LRH-116 and the estrogen receptor (Ref. 17), interaction with corepressors such as NCoR (Ref. 18) and Alien (Ref. 19), and binding to RNA and directing its shuttling between the nucleus and cytoplasm (Ref. 20). Loss of NR0B1 expression or function results in adrenal hypoplasia congenita, in which the fetal adrenal gland fails to undergo differentiation into the adult gland (Ref, 13). Duplication of the NR0B1 gene (with resulting increased expression) results in a sex-reversal phenotype (Ref. 13).

Using our newly-developed Ewing’s sarcoma-based model system, we verified that knock-down of EWS/FLI resulted in a decreased expression of NR0B1 (Ref. 10). We then found that knock-down of NR0B1 expression resulted in a nearly complete loss of oncogenic transformation (Ref. 10). This effect was specific to NR0B1 loss, and wasn’t an "off-target" effect of the RNAi, because oncogenic transformation was restored by re-expression of NR0B1, using a version that was immune to the RNAi construct (Ref. 10). Finally, using a series of over thirty different tumor cell lines, we showed that NR0B1 expression was specific for Ewing’s sarcoma (Ref. 10). These data suggest that NR0B1 may be a new therapeutic target for Ewing’s sarcoma.

Unfortunately, a drug targeting NR0B1 does not currently exist, nor do we have an understanding of the molecular mechanisms that NR0B1 uses to mediate oncogenic transformation in Ewing’s sarcoma. Our lab is now focused on understanding the function of NR0B1 in Ewing’s sarcoma. We recognize that various potential functional domains have been identified in NR0B1, such as a putative ligand binding domain, transcriptional repression domains, and LXXLL protein binding motifs. We are in the process of determining which of these domains are required for NR0B1 to participate in oncogenic transformation. Furthermore, we have identified a number of potential protein binding partners of NR0B1 in Ewing’s sarcoma cells. We hope that by defining key domains and protein partners of NR0B1, we will develop a better understanding of NR0B1 function in Ewing’s sarcoma. This may then allow us to identify new approaches that might functionally inhibit NR0B1, and serve as a new therapeutic direction for Ewing’s sarcoma.

Summary and conclusions

Ewing’s sarcoma remains an enigmatic tumor. Through a combination of better diagnostic and prognostic approaches and new therapeutic agents, we believe that great opportunities exist for improving the care of patients with this disease. While much work is left to be done, the foundation is firmly placed, and can now be used to build the crucial molecular understanding that is required to cure this devastating illness.

Editor's Update: Huntsman Cancer Institute released a news story about this study on August 31, 2009. The study was funded, in part, by a $100,000 grant from the Liddy Shriver Sarcoma Initiative, which was credited in the article entitled, "CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis." The Initiative also funded previous research on EWS/FLI at Huntsman Cancer Institute.

References

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