Rap 1b as a mitogenic signal in thyroid

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） 目前还不清楚它们是如何从单一的终末分化细胞类型中产生的，或者它们是否 具有单克隆或多克隆来源。我们最近对甲状腺干细胞的鉴定和体外培养 干细胞-甲状腺细胞分化程序的特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来自具有一定自我更新的干/祖细胞 特性. TSH，通过cAMP，是甲状腺增殖的关键成分，涉及PKA依赖性和 独立事件;然而，TSH-cAMP作用的主要效应物仍然难以捉摸。激动剂 增加细胞内cAMP能够快速磷酸化并激活Rap1b，我们最近已经证明 Rap-GEF Epac与PKA协同作用是TSH依赖性有丝分裂所必需的。与此相一致， 甲状腺中组成型活性G12V-Rap1b表达触发致甲状腺肿（即TSH-cAMP）- 依赖性肿瘤发生表型。 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， 在甲状腺肿形成时招募的其中，Sca 1+干细胞群体最近被鉴定。因为Rap1是 已知在几种细胞类型中调节促炎因子和趋化因子的释放， 最近证实在皮肤癌发生中TPA介导的炎症阶段是至关重要的，我们 假设致甲状腺肿的治疗在甲状腺中触发了由甲状腺激素介导的炎症样反应。 肿瘤发生过程所需的甲状腺外细胞的rap1依赖性募集。 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。 该系统依赖于其产生随机双等位基因分布、红色或绿色荧光的能力，利用 CRE/Lox技术。不可逆地标记特定细胞类型的能力允许谱系追踪实验 以监测荧光红色标记的细胞向绿色荧光室的募集/归巢。这 本提案将开发一条新的线来评估上述与肿瘤相关的未解决问题 克隆性和骨髓来源的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新的机制， 甲状腺肿

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 extrathyroideal 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 extrathyroideal 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.