Role of YB-1 in EGFR regulation and breast cancer

描述(申请人提供)：表皮生长因子受体(EGFR)的异常表达是乳腺癌预后不良的指标。然而，EGFR升高的机制尚不清楚。在基因筛查中，我们分离了YB-1(Y盒结合蛋白1)，一种多功能的基因转录和翻译调节因子，以确定介导EGF非依赖性乳腺癌细胞增殖的基因。我们发现YB-1在永生的人乳腺上皮细胞(HMECs)中的过度表达可以使EGF独立并增加EGFR水平。YB-1与EGFR基因控制区结合，乳腺癌标本中YB-1水平升高与EGFR表达及患者预后不良相关。此外，YB-1转基因小鼠会患上乳腺肿瘤。有趣的是，Akt使Ser102上的YB-1磷酸化，这是YB-1核定位、EGFR诱导和生长刺激所必需的残基。我们假设YB-1和EGFR是一个正反馈环路的一部分，在这个环路中，EGFR信号增加了YB-1的核定位；核YB-1结合到启动子区域，调节EGFR和其他控制增殖的基因的转录，从而促进乳腺癌的形成。1)研究YB-1对人脐静脉内皮细胞中EGFR的转录调控。我们将使用染色质免疫沉淀、报告分析和电泳迁移率分析来分析EGFR基因中介导YB-1诱导的功能性YB-1结合元件。2)探讨YB-1刺激EGF非依赖性细胞生长的机制。我们将确定：(A)YB-1诱导的乳腺上皮细胞EGF非依赖性是否需要YB-1 Ser102的磷酸化和核转位；(B)YB-1激活EGFR信号通路；(C)YB-1在HMEC中的敲除逆转EGF非依赖性，并触发细胞周期停滞、衰老或凋亡；(D)YB-1诱导的EGF非依赖性需要EGFR的诱导和负生长调节因子的抑制。3)检测YB-1和EGFR在肿瘤形成中的作用。我们将确定在人乳腺癌细胞中靶向YB-1和/或EGFR是否会减少它们在体外的锚定非依赖性生长以及它们在裸鼠原位模型中的致瘤和转移潜能。拟议的研究将加深我们对乳腺癌发展方式的理解。最重要的是，这项工作将研究在肿瘤中靶向YB-1和EGFR是否代表了治疗目前治疗选择有限的乳腺癌的一个子集的可行策略。

DESCRIPTION (provided by applicant): Aberrant expression of the epidermal growth factor receptor (EGFR) is an indicator of poor prognosis in breast cancer. However, the mechanism by which EGFR becomes elevated is unclear. In a genetic screen to identify genes that mediate EGF-independent proliferation of breast cancer cells, we isolated YB-1 (Y-box binding protein 1), a multifunctional regulator of gene transcription and translation. We found that YB-1 overexpression in immortal human mammary epithelial cells (HMECs) conferred EGF independence and increased EGFR levels. YB 1 binds to the EGFR gene control regions, and elevated YB-1 levels in breast cancer samples correlate with EGFR expression and with poor patient outcome. Moreover, YB 1 transgenic mice develop mammary tumors. Interestingly, Akt phosphorylates YB-1 on Ser102, a residue necessary for YB-1 nuclear localization, EGFR induction, and growth stimulation. We hypothesize that YB-1 and EGFR are part of a positive feedback loop whereby EGFR signaling increases YB-1 nuclear localization; nuclear YB-1 binds to promoter regions to regulate transcription of EGFR and other genes controlling proliferation, which contributes to breast cancer formation. We propose the following Specific Aims: 1) Investigate the transcriptional regulation of EGFR by YB-1 in HMECs. We will analyze functional YB-1 binding elements in the EGFR gene that mediate induction by YB-1 using chromatin immunoprecipitation, reporter assays, and electrophoretic mobility assays. 2) Investigate the mechanism by which YB-1 stimulates EGF-independent cell growth. We will determine if (a) YB-1-induced EGF independence of breast epithelial cells requires YB-1 Ser102 phosphorylation and nuclear translocation; (b) YB-1 activates EGFR signaling pathways; (c) knockdown of YB-1 in HMECs reverses EGF independence and triggers cell cycle arrest, senescence or apoptosis; (d) YB-1-induced EGF independence requires both induction of EGFR and repression of negative growth regulators. 3) Examine the role of YB-1 and EGFR in tumor formation. We will determine if targeting YB-1 and/or EGFR in human breast cancer cells reduces their anchorage-independent growth in vitro and their tumorigenic and metastatic potential in an orthotopic nude mouse model. The proposed studies will deepen our understanding of the way breast cancer develops. Most importantly, this work will examine whether targeting YB-1 and EGFR in tumors represents a viable strategy for therapy in a subset of breast cancer where treatment options are currently limited.