Cofilins通过YAP/TAZ介导甲状腺癌肿瘤干细胞干性转化的机制研究

Risistance to conventional radiotherapy and chemotherapy, and consequently high frequency of metastasis and relapse is the main reason for the poor prognosis of refractory thyroid cancer. Cancer stem cell (CSC) theory suggests that targeting CSCs may cure refractory thyroid cancer. Our previous study showed that thyroid CSCs expressed less Cofilins compared with non-CSCs and knockout Cofilin-1 can significantly increase the proliferation of thyroid cancer cells. Cofilin signaling pathway was closely related to the stemness transformation. Deletion of Cofilins can activate YAP/TAZ and reprogram non-stem cells into cells with full stem cell attributes. Based on the previous studies, we proposed the hypothesis that cofilins may regulated stemness transformation of thyroid CSCs via YAP/TAZ. We will use CRISPR / Cas9 technology to knockout Cofilins or generate mutations of Cofilins in thyroid cancer cells, and compare their phenotypes (including their potency of self renewal and differentiation, epithelial interstitial transformation and iodine intake rate) in vitro and in vivo. We will try to find out if knockout Cofilins can induce activation of YAP/TAZ by mechanical transduction signal and promote the expression of target genes, which may result in stemness transformation of tumor cells. If succeed, we will elucidate the role and mechanism of Cofilins in the stemness transformation of thyroid CSCs, and lay out the foundation for the development of CSC targeting therapy for refractory thyroid cancer.

对放化疗不敏感且易复发转移是难治性甲状腺癌预后差的主要原因，靶向肿瘤干细胞（CSC）的治疗有望治愈难治性甲状腺癌。我们前期研究发现甲状腺癌CSC较非CSC的Cofilins表达下调；敲除Cofilin-1可使甲状腺癌细胞增殖能力明显增强；Cofilins与正常细胞的干性转化关系密切，其机制与YAP/TAZ相关；据此提出Cofilins可能通过YAP/TAZ介导甲状腺CSC干性转化。本课题拟采用CRISPR/Cas9技术敲除甲状腺癌细胞中的Cofilins或引入其突变，通过体内外实验进行细胞表型比较（包括自我更新及分化潜能，EMT，摄碘率等），探索敲除Cofilins可否通过机械转导信号使YAP/TAZ激活促进靶基因的表达而使甲状腺CSC干性激活，阐明Cofilins在甲状腺CSC干性转化中的作用及机制，为研发难治性甲状腺癌的CSC靶向治疗奠定基础。

难治性甲状腺癌是目前甲状腺癌治疗的难点，也是近年来我国甲状腺癌死亡率年均增长1.6%的主要原因。本课题组通过Meta分析及临床回顾性研究明确了与难治性甲状腺癌淋巴结转移密切相关的危险因素。接着构建特异性的cofilin的FRET生物探针，但是反复尝试通过CRISPR/Cas9基因编辑技术敲除细胞内Cofilins（ADF-Cofilin和／或Cofilin-1）同时敲入定点突变的ADF-CofilinS3A和／或Cofilin-1S3A，建立CofilinS3A的甲状腺癌细胞系均不成功，于是我们转向了难治性甲状腺癌的CAR-T药物的研究。我们以目前临床应用最为广泛的第二代CAR结构为基础，设计了靶向TSHR的CAR，其结构包括靶向TSHR的scFv分子和来源于CD28的共刺激分子以及胞内的CD3ζ信号激活结构域。接着我们将不同scFv设计的anti-TSHR CAR基因转导入病毒包装细胞，通过病毒感染将CAR导入分离提取了人的外周血淋巴细胞T细胞，制成Anti-TSHR CAR-T细胞。我们通过体外杀伤实验显示K70-H和Kab-H两种CAR对靶细胞有较好的细胞毒性。下一步我们拟通过体内实验验证针对我们研制的anti-TSHR CAR-T细胞的功能和特异性并进行优化。本项目如获成功，针对CAR-T治疗复发转移型PTC若获得初步的体外安全性和有效性结论，这将为进一步的药物研发提供支持，有助于推动CAR-T药物尽快推向市场，有望代替手术治疗可手术的复发转移的PTC及实现对无法手术的复发转移的难治性PTC的治疗突破。