Role of Notch 1 signaling in esophageal carcinogenesis

Notch 1信号在食管癌发生中的作用

• 批准号: 8456343
• 负责人: Kelly A Whelan
• 金额: $ 4.92万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8456343
• 关键词: 3-Dimensional; Biological Assay; Biology; CD44 gene; Cell Differentiation process; Cell Line; Cells; Characteristics; Data; Development; Diagnosis; Differentiation Antigens; Disease; Disease Progression; Engineering; Epithelial; Esophageal; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Foundations; Gene Targeting; Generations; Genetically Engineered Mouse; Human; Hyaluronic Acid; Hyperplasia; Immunoprecipitation; In Vitro; Lesion; Maintenance; Malignant - descriptor; Malignant Epithelial Cell; Malignant Neoplasms; Mediating; Mesenchymal; Migration Assay; Molecular; Mus; Oncogenes; Pathway interactions; Patients; Population; Property; Publishing; Reporting; Research; Resistance; Role; Scientific Advances and Accomplishments; Signal Pathway; Signal Transduction; Skin; Squamous Cell; Squamous Differentiation; Squamous cell carcinoma; System; Testing; Transcriptional Regulation; Transforming Growth Factor beta; Transforming Growth Factors; Tumor Biology; Tumor Suppressor Proteins; Tumorigenicity; base; cancer cell; cancer therapy; cancer type; carcinogenesis; chemoradiation; chromatin immunoprecipitation; human TGFB1 protein; in vivo; innovation; insight; keratinocyte; lentiviral-mediated; monolayer; mouse model; neoplastic cell; notch protein; novel; novel strategies; novel therapeutics; outcome forecast; overexpression; promoter; public health relevance; receptor; receptor-mediated signaling; response; self-renewal; therapy development; tumor; tumor growth; tumor initiation; tumor progression; tumor xenograft; tumorigenesis; tumorigenic

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.

描述（由申请人提供）：食管鳞状细胞癌（ESCC）是人类鳞状细胞癌（SCC）最具侵袭性的形式之一。 ESCC患者的预后仍然很差，由于晚期诊断和有限的反应，放化疗，强调需要新的治疗策略，在治疗这种疾病。 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学抗性的肿瘤细胞的子集，这些特性有助于它们作为癌症治疗靶点的出现。透明质酸受体CD 44高表达的TIC已在ESCC中被鉴定，然而，调节这些亚群的机制尚未阐明。我们已经从ESCC细胞中分离出显示TIC特征的CD 44高（CD 44 H）群体，并证明转化生长因子（TGF）-β促进CD 44 H亚群的扩增，同时激活Notch信号传导。Notch通路是细胞命运的环境依赖性调节因子，在多种癌症类型中失调，然而，该通路在ESCC中的作用尚不清楚。在目前的建议中，我们假设在ESCC侵袭性前沿的Notch（N1）激活与TGF-β协同作用，以促进ESCC的扩张。 CD 44 H细胞群与ESCC的恶性特性相关。我们将通过三个相互关联的具体目标来检验这一点。 目的1：阐明N1在CD 44 H ESCC细胞扩增中的作用。这将使用诱导型慢病毒过表达和沉默系统来实现，以确定在单层和三维培养中产生CD 44 H细胞时对N1信号传导的需求。这些表达系统也将与迁移、侵袭、集落形成和异种移植肿瘤形成的测定一起使用，以评估N1在CD 44 H细胞介导的致瘤性中的作用。目的2是表征N1和TGF-β协同促进CD 44 H细胞扩增的分子机制。 基于已发表的研究结果和初步数据，我们假设TGF-β和N1介导的信号通路可能相互干扰，从而启动涉及抑制N1靶基因Notch 3（N3）的细胞命运转换。我们将通过使用免疫沉淀（IP），启动子活性测定和染色质IP检查TGF-β对N1介导的N3转录调控的影响来测试这一点。 我们还将使用诱导型慢病毒介导的过表达和沉默系统来研究N3信号在CD 44 H亚群产生中的作用。目的3是使用基因工程小鼠模型来表征N1在体内肿瘤生长和TIC扩增中的作用。我们将通过使用诱导型K5-ERT-Cre~ Notch 1敲除/lox小鼠灭活已建立的ESCC病变中的N1并评估对肿瘤进展和TIC含量的影响来评估N1在ESCC进展中的作用。总的来说，这些研究将揭示N1支持ESCC进展的机制，同时为ESCC治疗的翻译应用建立新的途径平台，ESCC对现有疗法仍然具有抗性。