Rap 1b as a mitogenic signal in thyroid

描述(申请人提供)：甲状腺肿瘤是人类最常见的内分泌恶性肿瘤。甲状腺细胞来源的肿瘤有不同的类型(滤泡性腺瘤、乳头状癌、滤泡性癌和间变性癌，以及它们的变体)，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，也不清楚它们是由单克隆性还是多克隆性起源的。我们最近对甲状腺干细胞的鉴定和对干细胞-甲状腺细胞分化计划的体外鉴定将使我们能够解决甲状腺肿瘤是源于终末甲状腺细胞的去分化还是源于具有某些自我更新特性的干细胞/祖细胞。TSH通过cAMP是甲状腺增殖的关键成分，既涉及PKA依赖事件，也涉及独立事件；然而，TSH-cAMP作用的主要效应分子仍不清楚。增加细胞内cAMP的激动剂能够快速磷酸化和激活Rap1b，我们最近表明，Rap-Global EPAC与PKA协同作用，是TSH依赖的有丝分裂发生所必需的。与此一致的是，G12V-Rap1b在甲状腺中的结构性活性表达触发了甲状腺激素(即TSH-cAMP)依赖的致瘤表型。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生成时招募的。其中，最近发现了一批Sca1+干细胞。由于已知Rap1在几种细胞类型中调节致炎和趋化因子的释放，并且最近被证明在TPA介导的皮肤癌的炎症阶段起关键作用，我们假设促甲状腺激素治疗在甲状腺中触发炎症样反应，介导的炎症反应是肿瘤发生过程中所需的依赖于Rap1的甲状腺外细胞的募集。我们开发了一种新的报告线，可用于克隆性确定和血统追踪研究。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。不可逆地标记特定细胞类型的能力允许进行谱系追踪实验，以监控荧光红色标记细胞进入绿色荧光隔间的招募/归巢。这项建议将利用这一新的线来评估上述与肿瘤克隆性相关的悬而未决的问题，以及骨髓来源细胞在甲状腺肿瘤发生过程中的潜在作用。拟议研究的成功完成将为甲状腺生成过程提供新的机制。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。甲状腺结节转化是甲状腺肿大的一个标志。近20%的人口(女性患病率)在一生中会出现甲状腺结节。然而，其中涉及的机制尚不清楚。最初被认为是针对终末分化的甲状腺细胞的累积突变过程，目前的模型认为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，如果能开发出合适的模型的话。我们已经开发了一种新的策略来解决其中的一些问题，这应该会为复杂的肿瘤发生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.