Role of Notch 1 signaling in esophageal carcinogenesis

描述(申请人提供)：食道鳞状细胞癌(ESCC)是人类鳞状细胞癌(SCC)中最具侵袭性的形式之一。由于诊断较晚和放化疗反应有限，食管鳞癌患者的预后仍然很差，这突显了在这种疾病的治疗中需要新的治疗策略。肿瘤起始细胞(TICs)是具有自我更新和分化能力以及增强的化疗耐药性的肿瘤细胞亚群，这些特性有助于它们成为癌症治疗的靶点。在食管鳞癌中已发现由透明质酸受体CD44的高表达所定义的抽动，但调控这些亚群的机制尚不清楚。我们已经从ESCC细胞中分离出具有TICS特征的CD44High(CD44H)亚群，并证明转化生长因子(TGF)-β在激活Notch信号的同时促进CD44H亚群的扩张。Notch通路是细胞命运的上下文依赖性调节因子，在不同的癌症类型中调节失调，然而，该通路在ESCC中的作用尚不清楚。在目前的提议中，我们假设在ESCC侵袭性前沿的Notch(N1)激活与转化生长因子-β协同作用，以促进 与ESCC恶性相关的CD44H细胞群。我们将通过三个相互关联的具体目标来测试这一点。目的1阐明N1在CD44H ESCC细胞群扩增中的作用。这将使用可诱导的慢病毒过表达和沉默系统来确定在单层和三维培养中产生CD44H细胞时对N1信号的需求。这些表达系统还将与迁移、侵袭、克隆形成和异种移植瘤形成的检测相结合，以评估N1在CD44H细胞介导的肿瘤形成中的作用。目的2研究N1和转化生长因子β协同促进CD44H细胞增殖的分子机制。根据已发表的发现和初步数据，我们假设转化生长因子-β和N1介导的信号通路可能相互干扰，启动细胞命运转换，涉及抑制N1靶基因Notch 3(N3)。我们将通过免疫沉淀(IP)、启动子活性分析和染色质IP检测转化生长因子-β对N1介导的N3转录调控的影响来检验这一点。我们还将使用可诱导的慢病毒介导的过表达和沉默系统来研究N3信号在CD44H亚群产生中的作用。目的3是利用基因工程小鼠模型研究N1在体内肿瘤生长和TIC扩张中的作用。我们将通过使用可诱导的K5-ERT-Cre~Notch1flxf/lox小鼠在已建立的ESCC病变中灭活N1来评估N1在ESCC进展中的作用，并评估对肿瘤进展和TIC含量的影响。总而言之，这些研究将揭示N1支持ESCC进展的机制，同时为翻译应用于ESCC治疗的新途径建立一个平台，ESCC仍然对现有的治疗方法具有抵抗力。

DESCRIPTION (provided by applicant): Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of human squamous cell carcinoma (SCC). ESCC patient prognosis remains poor due to late diagnosis and limited response to chemoradiotherapy, highlighting the need for novel therapeutic strategies in the treatmen of this disease. Tumor-initiating cells (TICs) are subsets of tumor cells possessing self-renewal and differentiation capabilities as well as enhanced chemoresistance, properties that have contributed to their emergence as cancer therapy targets. TICs, defined by high expression of CD44, the receptor for hyaluronic acid, have been identified in ESCC~ however, mechanisms regulating these subpopulations have yet to be elucidated. We have isolated CD44 High (CD44H) populations displaying characteristics of TICs from ESCC cells and demonstrated that transforming growth factor (TGF)-beta, promotes expansion of CD44H subpopulations concomitant with activation of Notch signaling. The Notch pathway is a context-dependent regulator of cell fate that is dysregulated in diverse cancer types~ however, the role of this pathway in ESCC is not yet known. In the current proposal, we hypothesize that Notch (N1) activation in the invasive front of ESCC acts in concert with TGF-beta to facilitate expansion of CD44H cell populations that are associated with the malignant properties of ESCC. We will test this through three interrelated specific aims. Aim 1 is to elucidate the role of N1 in expansion of CD44H ESCC cell populations. This will be achieved using inducible lentiviral overexpression and silencing systems to determine the requirement for N1 signaling in generation of CD44H cells in monolayer and 3-dimensional culture. These expression systems will also be used in concert with assays of migration, invasion, colony formation and xenograft tumor formation to assess the role of N1 in CD44H cell- mediated tumorigenicity. Aim 2 is to characterize the molecular mechanisms through which N1 and TGF- beta cooperate to promote CD44H cell expansion. Based on published findings and preliminary data, we hypothesize that TGF-beta- and N1-mediated signaling pathways may crosstalk to initiate a cell fate switch involving suppression of the N1 target gene Notch 3 (N3). We will test this by examining the influence of TGF-beta on N1-mediated transcriptional regulation of N3 using immunoprecipitation (IP), promoter activity assays and chromatin IP. We will also use inducible lentiviral-mediated overexpression and silencing systems to investigate the role o N3 signaling in generation of CD44H subpopulations. Aim 3 is to characterize the role of N1 in tumor growth and TIC expansion in vivo using genetically engineered mouse models. We will evaluate the role of N1 in ESCC progression by inactivating N1 in established ESCC lesions using inducible K5-ERT-Cre~Notch1floxf/lox mice and assessing effects on tumor progression and TIC content. Collectively, these studies will reveal mechanisms through which N1 supports ESCC progression while building a platform for new avenues toward translational applications for therapy of ESCC, which remains resistant to existing therapies.