TSHR信号通路对分化型甲状腺癌失分化的影响机制研究

Thyroid cancer is the most common endocrine malignant tumor and the majority is differentiated thyroid carcinomas (DTC). The metastases of DTC can retain their ability to concentrate iodine, so radioiodine (131I) therapy is effective. However, about one third of thyroid cancer metastases can lose their ability to concentrate 131I, which indicates a poor prognosis. NIS, the key molecule responsible for the iodide concentration, has been frequently studied. Some techniques have been tried to regulate NIS expression, in order to enhance or reestablish 131I uptake. These methods utilize agents such as retinoic and NIS gene transfection, but the results have not been encouraging. TSHR molecules in the membrane are quite stable and signaling in the thyrocyte is controlled mainly by circulating TSH levels. T4 withdrawal or TSH administration to DTC patients with functional TSHR ensures uptake of 131I and the removal of malignant cells. However, some metastases may have no radioiodine uptake even if circulating TSH is high. We have proved previously that FTC-133 cells underwent dedifferentiation with decreasing of TSHR expression. By TSHR gene transfection, 131I uptake of FTC-133 was enhanced. So, we hypothesized that TSHR signal anomalies may play a key role in the dedifferentiation of DTC. First, TSHR expression or function may be changed. A variety of factors can lead to decreased expression of TSHR, such as methylation of TSHR gene promoter and TSHR gene mutations. However, in thyroid cancer, few studies about the TSHR mutations have been reported. Then, TSH-TSHR signaling pathways may be abnormally activated. The combination of TSH and TSHR activate mainly four kinds of G protein family-Gs, Gi/o, G12/13 and Gq/11. The most important regulator of thyroid function is activated by cAMP pathway through Gs proteins. Gq/11 protein can activate PLC-PI3K-Ca2+ pathway, playing an important role in cell proliferation and tumorigenesis. Accordingly, this study will detect TSHR expression and function of different thyroid cancer cell lines, including TSHR gene mutation and methylation status of the promoter. Then, we will study the impact of activation of different G protein on iodine uptake of thyroid cancer cell lines. Furthermore, crosstalk of MAPK, PI3K pathway and TSHR pathways and their combining effect on iodione uptake of thyroid cancer will be investigated. At last, TSHR with activating mutation will be transfected into thyroid cancer cell lines to determine iodine uptake and apoptosis of thyroid cancer cells. This project will further clarify the physiological function of TSHR and illuminate the role of TSHR signaling pathway in dedifferentiation of thyroid cancer. And furthermore specific mutation of TSHR with activation of Gs protein genes tansfected into the cells will improve of the iodine uptake of the cells, which would provide a novel way to treat dedifferentiated thyroid cancer effectively.

分化型甲状腺癌失分化而出现摄碘功能障碍是制约131I治疗效果并导致其预后变差的主要原因。通过上调NIS蛋白以促进甲状腺癌细胞摄碘的方法效果欠佳，而从MAPK、PI3K等信号通路途径的角度亦未能明确阐述失分化发生的机制。本小组在前期工作中发现TSHR的表达异常影响甲状腺癌细胞株摄碘，结合国内外最新研究成果，我们设想TSHR信号通路异常可能在甲状腺癌失分化过程中扮演非常重要的角色。本项目从通过对TSHR基因表达和蛋白功能的检测，TSH-TSHR通路的下游信号的改变，包括四种G蛋白亚基激活进行一系列研究，以期发现TSHR蛋白表达及信号通路对甲状腺癌失分化的影响，并深入研究TSHR通路与BRAF、PI3K通路之间的相互关系，同时将激活型TSHR基因转染入甲状腺癌细胞，观察细胞摄碘及131I治疗疗效改善情况。最终，从新的角度阐述分化型甲状腺癌失分化的分子机制，并探寻有效的使其再分化的方法。

分化型甲状腺癌失分化而失去摄碘功能，使I-131治疗无效，并导致其预后变差，而其机制尚未明确。通过上调NIS蛋白以促进甲状腺癌细胞摄碘的方法效果不佳，而单纯从MAPK、PI3K等信号通路途径的角度亦未能明确阐述失分化发生的机制。在此项目中，本小组使用各种分化成度不同的甲状腺癌细胞株，包括甲状腺乳头状癌细胞株TPC1、甲状腺滤泡状癌细胞株FTC-133、低分化甲状腺乳头状癌细胞株BCPAP和未分化甲状腺癌细胞株8505C等，通过对其TSH-TSHR信号通路、NIS等甲状腺摄碘蛋白、细胞株的摄碘功能、细胞株的转移和侵袭能力等的研究，并将野生型和突变型的TSHR转染入甲状腺癌细胞进一步观察上述各指标。研究发现，甲状腺癌细胞株的摄碘能力和侵袭性和TSHR的表达以及TSHR信号通路的异常激活相关。TSHR的低表达和非经典的G12/13、Gq/11等G蛋白的异常激活，影响甲状腺癌细胞株NIS等甲状腺特异蛋白的表达和细胞内异常定位，并影响的摄碘能力，同时增加RhoGTPase而增加细胞转移能力和侵袭能力。这一过程并与PI3K信号通路相互作用，进一步影响NIS等蛋白的细胞内定位和增加细胞的侵袭能力。本项目首次发现TSH-TSHR非经典信号通路的异常激活，一方面导致甲状腺分化蛋白，如NIS、Tg、TPO等的表达下降影响细胞摄碘，在甲状腺癌失分化中扮演者极其重要的作用，另一方面TSH-TSHR非经典通路的异常激活同PI3K/Akt/mTor信号通路相互作用，进一步增加细胞转移和侵袭能力，一方面回答了甲状腺癌失分化的原因，另一方面还回答失分化甲状腺癌预后下降的原因。同时指出，单纯将NIS导入甲状腺细胞中以再分化治疗，但是如果NIS 无法定位至细胞表面，不但无法解决甲状腺摄碘问题，反而将会进一步增加细胞的侵袭性。 针对TSHR的治疗将为失分化甲状腺癌的治疗提供新的途径。