ROLE OF EDR3 IN NORMAL DEVELOPMENT AND TUMORIGENESIS

EDR3 在正常发育和肿瘤发生中的作用

• 批准号: 7116891
• 负责人: MARC F HANSEN
• 金额: $ 31.48万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-29 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7116891
• 关键词: 3T3 cells; carcinogenesis; cell differentiation; cell growth regulation; clinical research; flow cytometry; gel mobility shift assay; gene deletion mutation; human genetic material tag; human tissue; immunofluorescence technique; immunoprecipitation; mass spectrometry; mesenchyme; neoplasm /cancer genetics; northern blottings; osteoblasts; osteosarcoma; polymerase chain reaction; southern blotting; stem cells; tumor suppressor genes; western blottings

DESCRIPTION (provided by applicant): We identified Early Developmental Regulator 3 (EDR3), as a candidate tumor suppressor gene in a screen of a region of human chromosome 3q26 previously shown to undergo LoH in osteosarcoma. We found intragenic deletions in the EDR3 gene consistent with a tumor suppressor etiology in osteosarcoma primary tumors and tumor cell lines. Examination of the expression pattern of EDR3 revealed that both mRNA and protein showed a near ubiquitous pattern of expression in adult and embryonic tissues. EDR3 is a homolog of the Drosophila Polyhomeotic gene and a member of the Polycomb Group (PcG) family of proteins. The PcG family of proteins acts as long-term repressors by contributing to the formation and stable transmission of heterochromatin. By co-immunoprecipitation, we found that the EDR3 protein was bound to E2F6, Bmi1, YY1 and M33 but not pRB1 or CtBP. This suggested that EDR3 was part of a previously described human Polycomb repressive complex (hPRC-H), which contains E2F6 and is active in G0. Consistent with this observation, we found that as cells entered G0, EDR3 localization in the nucleus changed from a diffuse to a highly punctuate pattern. This shift coincided with the ability of the EDR3-containing complex to bind to YY1 and E2F DNA-binding sites and to the c-myc promoter. When we tested co-localization of EDR3 with E2F6 and Bmi1, we found that the shift to a punctuate pattern of localization as the cells entered G0, was shared by EDR3 and E2F6 but not Bmi1. We found that EDR3 was affected in >70% of osteosarcoma tumors. Examination of other tumors showed that EDR3 protein was absent in other tumors suggesting that this could be a checkpoint, which is commonly lost in tumorigenesis. Consistent with this model, we found that EDR3 could act as a growth suppressor when overexpressed in normal cells, or when inducibly expressed in tumor cells. Our hypothesis is that the EDR3 protein acts as part of a repressor complex to regulate long-term maintenance of G0 in developing osteoblasts and mesenchymal stem cells. A corollary hypothesis is that loss of EDR3 function in osteoblast progenitor cells could lead to a loss of the ability to maintain G0 and thus favor tumorigenesis. To test our hypotheses, we propose the following specific aims: 1) To examine the role of EDR3 in osteosarcoma tumorigenesis and osteoblastic differentiation; 2) To characterize the components of the PcG complex containing EDR3 and E2F6; and 3) To identify the targets of EDR3 regulation.

描述(申请人提供)：我们发现早期发育调节基因3(EDR3)是一个候选的肿瘤抑制基因，位于人类染色体3q26的一个区域，该区域先前被显示在骨肉瘤中发生杂合性缺失。我们发现，在骨肉瘤、原发肿瘤和肿瘤细胞系中，EDR3基因的基因内缺失与肿瘤抑制病因学一致。对EDR3表达模式的研究表明，EDR3的mRNA和蛋白在成体和胚胎组织中的表达模式几乎无处不在。EDR3是果蝇多核基因的同源基因，是多梳蛋白家族的成员。PcG家族蛋白通过促进异染色质的形成和稳定传递而发挥长期抑制作用。通过免疫共沉淀，我们发现EDR3蛋白与E2F6、Bmi1、YY1和M33结合，而不与pRB1或CtBP结合。这表明EDR3是先前描述的人类多梳抑制复合体(HPRC-H)的一部分，该复合体含有E2F6并在G0中活性。与此一致的是，我们发现，随着细胞进入G0，EDR3在细胞核中的定位从弥漫模式转变为高度点状。这一转变与含有EDR3的复合体与YY1和E2F DNA结合位点以及c-myc启动子的结合能力相吻合。当我们测试EDR3与E2F6和Bmi1的共定位时，我们发现当细胞进入G0时，EDR3和E2F6共有的定位模式转变为点状定位模式，但Bmi1不同。我们发现EDR3在70%的骨肉瘤中受到影响。对其他肿瘤的检查显示，EDR3蛋白在其他肿瘤中缺失，这表明这可能是一个检查点，而这一点在肿瘤发生过程中通常是丢失的。与这一模型一致，我们发现EDR3在正常细胞中过表达或在肿瘤细胞中诱导表达时可以作为生长抑制因子。我们的假设是，EDR3蛋白作为抑制复合体的一部分，在发育中的成骨细胞和间充质干细胞中调节G0的长期维持。一个推论是，成骨细胞前体细胞中EDR3功能的丧失可能会导致丧失维持G0的能力，从而有利于肿瘤的发生。为了验证我们的假设，我们提出了以下具体目标：1)研究EDR3在骨肉瘤发生和成骨细胞分化中的作用；2)鉴定含有EDR3和E2F6的PcG复合体的组成；以及3)确定EDR3调控的靶点。