Rap 1b as a mitogenic signal in thyroid

描述（由申请人提供）：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的类型（滤泡腺瘤、乳头状癌、滤泡癌和间变性癌及其变体），目前尚不清楚它们是如何从单一的终末分化细胞类型产生的，也不清楚它们是单克隆还是多克隆起源的。我们最近对甲状腺干细胞的鉴定和干细胞-甲状腺细胞分化程序的体外表征将使我们能够确定甲状腺肿瘤是由终末甲状腺细胞的去分化还是来自具有一些自我更新特性的干细胞/祖细胞。通过cAMP， TSH是甲状腺增生的关键组成部分，涉及pka依赖性和独立事件；然而，TSH-cAMP作用的主要影响因子仍然难以捉摸。增加细胞内cAMP的激动剂能够快速磷酸化并激活Rap1b，我们最近发现Rap1b - gef Epac与PKA协同作用是tsh依赖性有丝分裂发生所必需的。与此一致的是，甲状腺中组成型活性G12V-Rap1b的表达引发了甲状腺素（即TSH-cAMP）依赖性的致瘤表型。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，这些细胞是在甲状腺形成时招募的。其中，最近发现了一个Sca1+干细胞群体。由于已知Rap1可以调节几种细胞类型中促炎因子和趋化因子的释放，并且最近被证明对tpa介导的皮肤癌变炎症期至关重要，我们假设甲状腺肿形成治疗触发甲状腺炎症样反应，由肿瘤发生过程所需的Rap1依赖性甲状腺外细胞募集介导。我们开发了一种新的报告系，适用于克隆测定和谱系追踪研究。该系统依靠其利用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细胞的招募/归巢到绿色荧光室。本文将利用这一新细胞系来评估上述未解决的与肿瘤克隆性相关的问题，以及骨髓来源细胞在甲状腺肿瘤发生过程中的潜在作用。成功完成所提议的研究将提供涉及甲状腺形成过程的新机制。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。甲状腺结节化是甲状腺甲状腺肿形成的标志。近20%的人口（以女性为主）在其一生中会出现甲状腺结节。然而，所涉及的机制尚不清楚。最初认为这是一个针对终末分化甲状腺细胞的突变累积过程，目前的模型提出甲状腺转化是一种干细胞疾病。如果能够开发出合适的模型，这两种可选择的模型有不同的预测。我们已经开发了一种新的策略来解决这些问题，这应该为肿瘤发生的复杂过程提供新的机制方面。

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.