激酶抑制剂联合表观遗传学药物诱导甲状腺癌再分化的作用及机制

Radioiodine-refractoriness represents a currently major challenge in the 131I therapy of differentiated thyroid carcinoma (DTC). The constitutive activation of signaling pathways, including MAPK, PI3K-AKT-mTOR, etc, plays a vital role in tumorigenesis, silencing of various thyroid iodide-metabolizing genes and the presence of malignant clinicopathologic features, which have also been demonstrated to be associated with epigenetic changes. Continuously sponsored by the National Natural Science Foundation of China (No.30700187 and 81271609), our group has successively achieved promising findings in many basic and clinical researches. Given the encouraging data of our preliminary study recently, which initially showed increased radioiodine uptake induced by sodium/iodide symporter gene transfer and successful modulation of iodide- and glucose-handling gene expression regulated by kinase inhibitors, the present study is designed to investigate the synergistic redifferentiation effect of kinase inhibitor and epigenetic drug in a variety of authenticated thyroid carcinoma cell lines harboring known gene alterations in vitro and in vivo. The effect of such novel redifferentiation strategy as well as the association between the effect and molecular genotype will be evaluated on gene, protein and functional levels using RT-PCR, Western blot, immunofluorescence, radioisotope uptake/efflux, clonogenic assay, micro-SPECT, micro-PET, and radionuclide therapy. Our project also aims at revealing the underlying mechanism and laying a foundation for molecular imaging and gene analysis in the precise redifferentiation treatment of DTC.

放射性碘抵抗是分化型甲状腺癌（DTC）131I治疗领域亟需解决的主要难题。研究发现，MAPK、PI3K-AKT-mTOR等信号通路的激活、表观遗传学改变均与DTC的发生、碘代谢相关基因沉默、恶性临床病理特征密切相关。受国家自然科学基金（编号：30700187和81271609）连续资助，我研究小组陆续证实了钠/碘转运体在介导肿瘤细胞摄碘中的关键作用及激酶抑制剂对DTC碘和葡萄糖代谢的有效调控。在此基础上，本研究拟选取多种已知突变DTC细胞系，通过RT-PCR、Western blot、免疫荧光、放射性核素摄取/流出、克隆形成等体外实验和小动物SPECT、小动物PET、核素治疗等体内实验，在基因、蛋白和功能水平上全面评价激酶抑制剂和表观遗传学药物是否存在协同再分化作用并揭示其潜在机制，分析其效能与DTC分子分型之间是否存在相关性，为分子影像和基因检测指导DTC的精准再分化治疗奠定基础。

目的：BRAFV600E突变导致MAPK信号通路过度激活引起分化型甲状腺癌发生失分化导致肿瘤不摄碘的主要的原因之一。MAPK信号通路抑制剂诱导甲状腺癌分化提升Nis表达，促进放射碘摄取效能仍待提高。本研究旨在通过组蛋白去乙酰化酶抑制剂/组蛋白修饰酶抑制剂联合MAPK信号通路抑制剂增强甲状腺癌分化，提升Nis表达及介导肿瘤细胞对放射碘的摄取，并探究其潜在机制。.方法：利用组蛋白去乙酰化酶抑制剂（HDACi ，帕比司他）或组蛋白甲基转移酶抑制剂（HMTi，Tazemetostat）联合BRAF抑制剂（达拉非尼）或MEK抑制剂（司美替尼）对BRAFV600E突变细胞系（BCPAP、K1）及BRAF野生型细胞系（BHP 2-7、TPC-1）处理后，利用ChIP、qRT-PCR、Western Blot、免疫荧光、细胞放射碘摄取实验及细胞克隆形成等实验验证联合用药对甲状腺癌细胞再分化的影响。.结果：帕比司他联合MAPK抑制剂时较单独药物增加了BRAFV600E突变细胞系Nis基因启动子乙酰化水平，而BRAF野生型细胞系经联合用药处理后，Nis基因启动子组蛋白乙酰化水平较单独药物处理无明显变化。同样，Tazemetostat联合MAPK抑制剂时较单独用药时明显降低了BRAFV600E突变细胞系H3K27me3水平，促进了Nis基因的表达。另外，单独使用促甲状腺激素（TSH）无法提升肿瘤细胞上述效应，但对药物处理后的甲状腺癌细胞，额外加入TSH能够不同程度提升药物对甲状腺癌细胞的分化效果。.结论：组蛋白表观修饰（HDACi 或 HMTi）联合MAPKi提升分化疗效的新路径，为碘难治性甲状腺癌患者靶向分化治疗提供了新理论。