Rap 1b as a mitogenic signal in thyroid

描述（由申请方提供）：甲状腺肿瘤是人类最常见的内分泌恶性肿瘤。甲状腺细胞源性肿瘤表现为不同类型（滤泡性腺瘤、乳头状癌、滤泡性癌和间变性癌及其变体），目前尚不清楚它们如何从单一终末分化细胞类型产生，或者它们是否具有单克隆或多克隆起源。我们最近的甲状腺干细胞的鉴定和干甲状腺细胞分化程序的体外表征将使我们能够解决甲状腺肿瘤是否起源于终末甲状腺细胞的去分化或具有一些自我更新特性的干/祖细胞。TSH，通过cAMP，是甲状腺增殖的关键组成部分，涉及PKA依赖和独立的事件;然而，TSH-cAMP作用的主要效应物仍然难以捉摸。增加细胞内cAMP的激动剂能够快速磷酸化并激活Rap 1b，我们最近发现Rap-GEF Epac与PKA协同作用，是TSH依赖性有丝分裂所必需的。与此一致，甲状腺中组成型活性G12 V-Rap 1b表达触发了致甲状腺肿（即TSH-cAMP）依赖性致瘤表型。越来越多的证据支持骨髓和炎症在肿瘤发生中的积极作用;特别是在甲状腺模型中，我们最近证明了甲状腺外细胞的存在，这些细胞在甲状腺肿发生时被招募。其中，Sca 1+干细胞群体最近被鉴定。由于Rap 1是已知的调节释放促炎和趋化因子在几种细胞类型，它最近被证明是关键的TPA介导的炎症阶段在皮肤癌的发生，我们假设，致甲状腺肿治疗触发甲状腺炎症样反应介导的Rap 1依赖性募集甲状腺外细胞所需的肿瘤发生的过程。我们已经开发了一种新的报告线适合克隆性测定和谱系追踪研究。该系统依赖于其利用CRE/Lox技术产生随机双等位基因分布、红色或绿色荧光的能力。不可逆地标记特定细胞类型的能力允许谱系追踪实验监测荧光红色标记的细胞向绿色荧光区室的募集/归巢。本提案将利用这一新细胞系来评估上述与肿瘤克隆性相关的未解决问题以及骨髓源性细胞在甲状腺肿瘤发生过程中的潜在作用。成功完成拟议的研究将提供甲状腺肿发生过程中涉及的新机制。公共卫生相关性：甲状腺肿瘤是人类最常见的内分泌恶性肿瘤。甲状腺结节性转化是甲状腺肿发生的标志。近20%的人口（女性患病率）在其一生中会出现甲状腺结节。然而，所涉及的机制是未知的。最初被认为是针对终末分化甲状腺细胞的突变的累积过程，目前的模型提出甲状腺转化是一种干细胞疾病。这两个替代模型有不同的预测，如果适当的模型可以开发。我们已经开发了一种新的策略来解决这些问题，应该提供新的机制方面的复杂过程中的肿瘤。

DESCRIPTION (provided by applicant): Thyroid tumors represent the most common endocrine malignancy in humans. Thyrocyte-derived tumors present as distinct types (follicular adenoma, papillary, follicular and anaplastic carcinoma, and their variants) and it is not clear how they can be generated from a single terminally differentiated cell type, or whether they have a monoclonal or polyclonal origin. Our recent identification of thyroid stem cells and an in vitro characterization of the stem-thyrocyte differentiation program will allow us to address whether thyroid tumors originate by de-differentiation of a terminal thyrocyte or from stem/progenitor cells with some self-renewal properties. TSH, via cAMP, is a key component in thyroid proliferation, involving both PKA-dependent and independent events; however, the main effectors of TSH-cAMP action still remain elusive. Agonists that increase intracellular cAMP are able to rapidly phosphorylate and activate Rap1b, and we have recently shown that the Rap-GEF Epac, in synergy with PKA, is required for TSH-dependent mitogenesis. Consistent with this, constitutively active G12V-Rap1b expression in the thyroid gland triggered a goitrogen (i.e. TSH-cAMP)- dependent tumorigenic phenotype. Increasing evidence supports an active role for bone-marrow and inflammation in tumorigenesis; specifically in the thyroid model we have recently demonstrated the presence of extrathyroidal cells that are recruited upon goitrogenesis. Among them, a population of Sca1+ stem cells was recently identified. Since Rap1 is known to modulate the release of proinflammatory and chemoattractant factors in several cell types, and it was recently demonstrated to be critical for the TPA-mediated inflammatory phase in skin carcinogenesis, we hypothesized that goitrogenic treatment triggers in the thyroid an inflammatory-like response mediated by the Rap1-dependent recruitment of extrathyroidal cells required for the process of tumorigenesis. We have developed a novel reporter line amenable for clonality determination and lineage tracing studies. The system relies on its ability to generate a random biallelic distribution, red or green fluorescence, utilizing the CRE/Lox technology. The ability to irreversibly tag specific cell types allows for lineage-tracing experiments to monitor recruitment/homing of fluorescently red-labeled cells into a green fluorescent compartment. This new line will be exploited in this proposal to assess the above-mentioned unresolved issues related to tumor clonality and the potential role of bone-marrow derived cells in the process of thyroid tumorigenesis. Successful completion of the proposed studies will provide new mechanisms involved in the process of goitrogenesis. PUBLIC HEALTH RELEVANCE: Thyroid tumors are the most common endocrine malignancies in humans. Thyroid nodular transformation is a hallmark of thyroid goitrogenesis. Almost 20% of the population (with prevalence in women) will present thyroid nodules during their lifetime. However, the mechanisms involved are unknown. Originally thought as a cumulative process of mutations targeting the terminally differentiated thyroid cells, current models proposed that thyroid transformation is a stem-cell disease. These two alternative models have distinct predictions, if appropriate models could be developed. We have developed a novel strategy to address some of these issues that should provide new mechanistic aspects of the complex process of tumorigenesis.