Ewing Sarcoma

by Mafalda von Alvensleben

Ewing Sarcoma, though unbeknownst to most of the general population, is a type of pediatric cancer that affects the soft tissue or bone. Approximately 225 people in the United States are diagnosed with this disease each year, primarily those between the ages of ten and twenty, making it comprise about 1% of all pediatric cancers. Ewing Sarcoma is typically discovered via radio-imaging techniques and confirmed by a biopsy of the affected area. The cancer presents itself most commonly in the lungs and long bones of the body; 41% of tumors are located in a lower extremity, 26% pelvis, 16% chest wall, 9% upper extremity, 6% spine, 3% hand and foot,  2% skull, while around 15% develop tumors exclusively in soft tissue [1]. The treatment options for those afflicted with pediatric Ewing Sarcoma typically involve some combination of the following therapies: an aggressive chemotherapy regimen, lasting ideally nine months to a year, radiation, and surgery if the sight is operable. Chemotherapy drugs utilized include: vincristine, doxorubicin, cyclophosphamide, ifosfamide, and etoposide which are now most commonly used in the United States and Europe. The current standard is to give vincristine, doxorubicin, cyclophosphamide over a two day infusion period followed by a five day infusion of ifosfamide and etoposide [2]. A more recent development in the treatment of Ewing Sarcoma has been “dose intense” chemotherapy regimens, which have become an increasingly desirable mode of therapy as the ability to manage the side effects of this treatment has improved. As indicated by the name, dose intense chemotherapy involves administering higher doses of chemotherapy over a shorter time period, while managing the levels of toxicity.  “The more recent Children’s Oncology Group Trial (AEWS0031) has shown that an every-two-week interval compression regimen is superior to the every three week regimen in both event-free and overall survival: 76 versus 65 percent event free survival at 4 years (p=0.029), and 91 versus 85 percent overall survival at 4 years (p=0.026).” [3] Those diagnosed with metastatic disease constitute 15% of Ewing Sarcoma cases, making for the most adverse prognostic outcome. However in patients whose primary tumor is isolated to the lungs tend to fare better than their metastatic counterparts.  This is best demonstrated by the fact that those diagnosed with metastasized tumors have a survival rate of 30% compared to those diagnosed earlier, who have a survival rate of about 70%. The survival rates also differ depending on age, with the survival rate of non-metastatic cancers being 56% for teens age 16 to 19 [4]. This is thought to be a result of the current treatment protocol that becomes more difficult for the body to endure as people age. There is also a discrepancy between pediatric and geriatric protocol since young adults and teenagers do not truly fit into either category mentally or biologically. Physically, young adults are at their peak, often able to withstand high dosage chemotherapy treatments that are ideal for their particular type of cancer. However, teenagers fair worse statistically than many younger patients, leading many to point fingers at the lack of research for such age groups. Others have even suggested that there may be mental and social contributions to treatment tolerance, but there is a lack of substantive research to support this. These factors make it particularly difficult to decide a course of action for the most effective treatments. There are many other prognostic elements that may impact survival and event free statistics on a case by case basis.  Also, due to the small sample size, the statistics that have already been gathered may not be sufficient for researchers to develop new treatment options. However, with new clinical trials underway that target known markers of Ewing Sarcoma, we may be closer than ever to finding new methods of treatment for patients with the disease. In order to assess the significance of these new treatments, their strengths and weaknesses, and which Ewing’s patients they have the potential to benefit, an understanding of the biology of Ewing Sarcoma is critical.

The biology of Ewing Sarcoma is not fully understood, however recent data and research have helped identify some promising factors. The most distinctive feature of Ewing Sarcoma is the translocation of chromosome 11 and 22, resulting in a chimeric molecule fusing the amino terminal-encoding portion of the EWS gene to the carboxyl terminal DNA binding domain encoded by the FLI1 gene [5]. The FLI1 gene encodes for a transcription factor which contains ETS-DNA binding domain and is particularly involved in developmental processes like regulating genes involved in hematopoeisis and homeostasis [6]. Additionally, EWS encodes for a protein that is involved in gene expression, cell signaling, and RNA processing and transport. The protein includes an N-terminal transcriptional activation domain and a C-terminal RNA-binding domain which are regions of this gene involved in the translocation that causes tumorigenesis associated with Ewing Sarcoma [7]. Denny et al., demonstrated that alterations to the structure of transcription factors play a critical in neoplastic transformation, making the aforementioned translocations of even greater interest. Karyotypic analyses have shown that the t(11;22) (q24;q12) chromosomal translocation presents itself in 86% of both Ewing and primitive neuroectodermal tumors, suggesting that this rearrangement is necessary in order to form such malignancies. In 5% of cases, there is a presentation of ERG(21q22). ERG is, like FLI1, an ETS transcription factor gene, that when fused to the EWS (locus 22q12) leads to the formation of chimeric protein that drives the malignant phenotype by affecting both gene transcription and RNA splicing of specific downstream genes [8]. ERG encodes for a protein of the same name whose function is to regulate transcription, and is involved in various other translocations resulting in fusion gene products such as TMPSSR2-ERG and NDRG1-ERG in prostate cancer and FUS-ERG in acute myeloid leukemia in addition to its role in Ewing sarcoma [9]. These proteins bind directly to DNA sequences through their DNA binding regions typically located in carboxyl terminus, altering the expression of near by genes and also interact through protein-protein interactions at the amino terminus  with other transcription factors that serve to activate gene expression in particular targets [10].  Mechanically, the translocation joins the N-terminal activation region of the EWS locus to the C-terminal ETS binding domain of the FLI1 gene, ultimately replacing the original transcription activation domains of the FlI1 and EWS sequences.  “At the genomic level the t(11;22) breakpoints are relatively tightly clustered within a 8 kilobase (Kb) region in the EWS locus but the FLI1 breakpoints are dispersed over approximately 35 Kb [11]. This can result in different EWS and FLI1 exons being incorporated into the fusion genes found in EFT tumor specimens.” [12]  A paper written by Arvand et al. in 2001 found that there were 12 different EWS/FLI1 fusions described each with variable combinations of exons flanking the fusion point [13]. However it was later amended in 2012 paper stating that four distinct fusion types have been isolated and studied. Fusion type one presents itself most often in children under the age of 13, and is often accompanied by improved prognostic factors [14]. Despite being correlated to a more positive prognosis, it is unclear whether  the specific fusion type can truly be pinpointed as the cause of improved prognosis.

The result of aforementioned fusions in the primary stage has been responsible for chimeric proteins, identified as the ES and EWS proteins. These proteins found specifically in Ewing are important for both the specificity in pathogenesis as well as the maintenance of sarcoma that identifies the disease [15]. The combination of of the EWS transcriptional activator and the FLI1 DNA-binding domain, causes them to act as aberrant transcription factors, potent repressors, or to alter RNA processing [16]. These fusion proteins bind to DNA in a site-specific manner, and act as potent transcriptional modulators. They are usually used as activators to regulate oncogenesis, but also occasionally repress more genes than up-regulate [17]. Modification of either the EWS transcriptional domain or the ETS DNA- indication for their role in transcriptional activation relies on the EWS domain.  “Additionally, EWS/FLI1 engages in post-transcriptional processes: in vitro it behaves as an aberrant RNA-splicing factor by blocking U1C-, TASR-, and YB1-mediated splicing. EWS/FLI1 interferes with normal EWS in the recruitment of EWS-binding proteins, uncoupling gene transcription from RNA-splicing, and consequently causing malignant transformation in ES.” [18]

The EWS/FLI1 fusion joins the activating domain of EWS to the DNA binding domain of FLI1, making it a probable aberrant transcription factor [19].  Therefore, identifying the targets of this fusion is a lucrative area of study for therapeutic targets to treat Ewing Sarcoma. Through ectopic expression of EWS-FLI1 in heterologous cell types as well as small interfering RNA-mediated knockdown of the fusion in ES cell lines, studies were able to discover potential target genes for therapies. These genes were identified as insulin-like growth factor (IGF) binding protein 3, and Aurora A and B Kinases [20].  These genes were found to be both up and down regulators, further emphasizing the role of EWS-FLI as a transcriptional activator as well as repressor. Normally,  IGF1 acts as an activator of cell proliferation pathways and as an inhibitor of apoptosis. It is it’s role as an apoptotic inhibitor that points to IGF-1’s role in tumour growth. “ Insulin-like growth factor (IGF)-I and its main binding protein, IGFBP-3, modulate cell growth and survival, and are thought to be important in tumour development.” [21] Insulin-like growth factor binding protein 3 (IGFBP-3) is known to prevent the interaction of IGF-1 with its receptor (IGF-R1), ultimately inhibiting cell proliferation, and inducing apoptosis in many human cancer cells through IGF-1 dependant and independent mechanisms [22]. Yet how these proteins interact with and are impacted by the EWS-FLI1 fusion is not fully understood. A study done by Cironi et. al  looked at IGF1 as a common target gene for Ewing Sarcoma and observed that IGFBP 3 was induced in a non DNA binding domain-dependant manner in the EWS-FLI1 fusion. This is in contrast to a previous report which stated that IGFBP-3 was a direct target of EWS-FLI1 and is downregulated by this fusion protein. The mode of regulation that EWS-FLI1 imposes on IGFBP-3 is thought to be dependent on cell state differences. The upregulation was observed in primary cells at an early stage of cancer following expression and prior to transformation and tumor development, while the downregulation reflected late state tumor progression. Therefore, the up versus down regulation is dependant on the cell type, and never occurs in the same cell simultaneously [23]. Despite IGF’s promising attributes as a therapeutic target, recent phase II studies using monoclonal antibodies targeting IGF1R show limited response rates of ~10% for patients with recurrent or refractory Ewing Sarcoma [24]. Due to the dependence on cell state connected to tumor progression and expression of IGFBP-3, future work is being directed towards identifying predictive biomarkers associated with patients with ES who could benefit from this type of treatment or combination therapy with other targeted agents and IGFBP-3 may be better suited as a biomarker.  

The IGF pathway has been the primary focus of recent clinical trials, however the upregulation of both Aurora A and B kinase by the EWS-FLI1 fusion has made them a desirable target for potential therapeutic treatment [25]. Aurora A kinase acts as a key regulatory component of the p53/TP53 pathway, and is involved in checkpoint-response pathways critical for oncogenic transformation cells of by phosphorylating and stabilizing tumor protein p53 (p53/TP53). It is also necessary for the disassembly of cilia prior to mitosis [26]. Similarly, Aurora B kinase is a key regulator of mitosis functioning at the centromere aiding in formation and alignment of the mitotic spindle, separation of the centrosomes and finally in the process of cytokinesis at the end of mitosis [27]. Since Aurora A and B are both cell cycle regulated kinases overexpressed in several other cancers, they have been used as targets in preclinical testing. These tests revealed a maintained complete clinical response for an Aurora kinase A inhibitor in as ES xenograft model, and a phase II trial analyzing the effects of the same drug in pediatric leukemia and solid tumors, including ES, is currently underway [28]. Further trials are also focusing on utilizing small molecule inhibitors to block RNA helicase A, a protein involved in the regulation of transcription and splicing and has been found to bind to EWS-FLI1, ultimately enhancing transcriptional activity. Preclinical evaluations have shown that the RNA helicase A inhibitor has induced apoptosis in ES cells and reduces tumor growth in xenografts, suggesting that it may also be effective in clinical trials [29].

Though the specificity of the EWS-FLI1 fusion in ES genes suggest that cell context is critical for EWS-FLI1 oncogenesis, the Ewing cell of origin is presently unknown [30]. This has made it difficult to identifying specific target genes based off of the development of homologous cell systems. Nevertheless, the target genes identified thus far show a promising angle for personalized medicine. The IGFBP-3 regulation by the EWS-FLI1 for instance could be used as an indicator for the type of treatment needed on a more case by case basis versus a ubiquitous strategy for all patients. This is a popular trajectory for cancer treatment as a whole, and because of the variability in the way EWS-FlI1 affects target genes Ewing Sarcoma is ideal cancer for this type of treatment development. Ultimately, Ewing Sarcoma, being such a rare cancer with a small sample size, has been greatly understudied and further investigation is required. However, the research that has been done thus far has proven to be useful for the development of new treatments for Ewing Sarcoma patients and other types of cancers. Furthermore, Ewings research reflects a shift in treatment approach to personalized medicine, making it a disease studied with attitudes directed to the forefront of medical research. Ultimately, with appropriate optimism and good science, there is hope for progress in the treatment of not only Ewings, but all forms of cancer.

Acknowledgments

I would first like to thank my Biology teacher Gina Aumock who not only taught me how to read and write scientific research, but also to have the courage to write about something so close me. I was diagnosed with Ewing Sarcoma at age fifteen in my right femur, and though I have been in remission for the past three years, I was curious to know more about what happened in my own body. Mrs. Aumock served as a mentor, editor, and fellow researcher on this project and I cannot fully express how grateful I am to her for all of her help. I would also like to thank my oncologist Dr. Noah Federman at UCLA for his enthusiastic encouragement of my writing this paper as well as providing invaluable information in the form of research material, experience, and editing.

Work Cited

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27. AURKB Gene. “Aurora Kinase B.” Gene Cards Human Gene Data Base. Accessed February 2018. http://www.genecards.org/cgi-bin/carddisp.pl?gene=AURKB&keywords=Aurora,A,B,Kinases.
28. Jennifer L. Anderson, Christopher T. Denny, William D. Tap, and Noah Federman. “Pediatric Sarcomas: Translating Molecular Pathogenesis of Disease to Novel Therapeutic Possibilities.” Pediatric Research 72, no. 2 (2012): 112-21. Accessed January 2018. doi:10.1038/pr.2
29. Jennifer L. Anderson, Christopher T. Denny, William D. Tap, and Noah Federman. “Pediatric Sarcomas: Translating Molecular Pathogenesis of Disease to Novel Therapeutic Possibilities.” Pediatric Research 72, no. 2 (2012): 112-21. Accessed January 2018. doi:10.1038/pr.2
30. A.Prieur, F. Tirode, P. Cohen, and O. Delattre. “EWS/FLI-1 Silencing and Gene Profiling of Ewing Cells Reveal Downstream Oncogenic Pathways and a Crucial Role for Repression of Insulin-Like Growth Factor Binding Protein 3.” Molecular and Cellular Biology 24, no. 16 (2004): 7275-283. Accessed January 2018. doi:10.1128/mcb.24.16.7275-7283.2004.