The Role of Eya3 as a downstream target of EWS Fli1 in Ewings Sarcoma

描述(申请人提供)：尤文氏肉瘤是一种侵袭性的儿科骨和软组织癌。出现转移的患者和复发的患者从这种疾病中预后特别差。尤因肉瘤是由染色体易位引起的，该易位产生了EWS/FLI1融合蛋白。我们已经确定发育蛋白Eya3是EWS/FLI1的下游靶点。Eya3对DNA修复很重要，也被发现是与六个同源框蛋白家族的两部分转录因子复合体的一部分。Eya/Six复合体对发育很重要，但当发育完成后表达不当时，会促进其他癌症的许多致癌特性，包括增殖、生存和转移。然而，这种复合体在儿童肿瘤中的作用，特别是尤文氏肉瘤，以前从未被研究过。与在其他人类肿瘤中观察到的相似，抑制尤文肉瘤细胞中的Eya3对增殖和存活的影响不大。然而，我们发现了Eya3在尤文肉瘤中的一个新的作用，其中Eya3基因敲除显著增加了尤文肉瘤细胞对化疗的敏感性。这可能是由于最近描述的Eya3促进有效DNA修复的能力的结果。此外，由于肿瘤起始细胞(TIC)与治疗耐药(化疗耐药)癌症有关，而且由于Eya3的结合伙伴SIX1增加了乳腺癌中的TIC群体，我们进一步研究了Eya3是否可能调节尤文氏肉瘤中的TIC群体。事实上，Eya3基因敲除减少了肿瘤启动细胞的数量。这一数据表明，Eya3可能通过多种可能相互关联的机制，在介导与复发疾病相关的尤文氏肉瘤表型中发挥重要作用。这项建议中概述的研究旨在更好地了解Eya3在尤文氏肉瘤复发相关表型中的作用，试图确定新的药物靶点，当被抑制时，可能会减少与尤文氏肉瘤相关的不良临床结果。所进行的研究将使我们能够确定EWS/FLI1调节Eya3的机制，然后进一步评估Eya3在调节肿瘤启动细胞群和化疗耐药中的作用。Eya3具有独特的酪氨酸磷酸酶结构域，以及与其致癌伙伴SIX1一起驱动转录所需的转录激活结构域。在这项提案中的工作将使我们能够确定Eya3的哪些活性对于其在尤文氏肉瘤中的作用是重要的，这样我们就可以更好地指导我们使用小分子抑制剂来靶向Eya3。

DESCRIPTION (provided by applicant): Ewing's sarcoma is an aggressive pediatric cancer of the bone and soft tissue. Patients that present with metastasis and patients who relapse have especially poor outcomes from this disease. Ewing's sarcoma is driven by a chromosomal translocation that produces the EWS/Fli1 fusion protein. We have identified the developmental protein, Eya3, as a downstream target of EWS/Fli1. Eya3 is important for DNA repair and is also found as part of a bipartite transcription factor complex with the Six family of homeobox proteins. The Eya/Six complex is important for development, but when inapropriately expresed after development is complete, promotes many oncogenic properties in other cancers, including proliferation, survival, and metastasis. However, the role of this complex in pediatric tumors, specificaly Ewing's sarcoma, has never before been examined. Similar to what is observed in other human tumors, inhibition of Eya3 in Ewing's sarcoma cells has a modest effect on proliferation and survival. However, we have discovered a novel role for Eya3 in Ewing's sarcoma, where Eya3 knockdown significantly increases Ewing's sarcoma cell chemosensitivity. This may be the result of the recently described ability of Eya3 to facilitate efficient DNA repair Additionally, since tumor-initiating cells (TIC) are implicated in treatment-resistant (chemoresistant) cancers, and since the binding partner of Eya3, Six1, increases TIC populations in breast cancer, we further examined whether Eya3 may modulate a TIC population in Ewing's sarcoma. Indeed, Eya3 knockdown decreases the tumor-initiating cell population. This data suggests that Eya3 may play an important role in mediating Ewing's sarcoma phenotypes associated with recurrent disease through multiple, and possibly inter- related, mechanisms. Studies outlined in this proposal aim to better understand the role of Eya3 in phenotypes associated with Ewing's sarcoma relapse, in an attempt to identify novel new drug targets that when inhibited, may reduce the poor clinical outcomes associated with Ewing's sarcoma. Studies performed will allow us to determine the mechanism of EWS/Fli1 regulation of Eya3 and then go on to further evaluate the role of Eya3 in modulating tumor-initiating cell populations and in chemoresistance. Eya3 has a unique tyrosine phophatase domain, as well as a transcriptional activation domain required to drive transcription along with its oncogenic partner, Six1. Work within this proposal will allow us to determine which activities of Eya3 are important for its roles in Ewing's sarcoma, so that we can better guide our targeting of Eya3 with small molecule inhibitors.