糖蛋白粘附分子VCAM1在甲状腺癌靶向BRAF治疗中的作用及机制研究

Refractory thyroid cancer is the major cause of death since no effective clinical treatment is available. BRAF mutation is the most important molecular alteration in thyroid cancer. BRAF inhibitor, Vemurafenib, has been applied to clinical treatment for melanoma. However, most thyroid cancer has low response to Vemurafenib and the mechanism is still unclear, which severely limits the clinical application. VCAM1 is a transmembrane glycoprotein belonging to immunoglobulin superfamily associated with metastasis, angiogenesis and treatment sensitivity of cancer. So far, the relationship of VCAM1 and BRAF inhibition has not been reported. Our preliminary study found that VCAM1 was obviously up-regulated during BRAF inhibition in thyroid cancer cells, and knock-down of VCAM1 increased the treatment efficacy. Besides, we found VCAM1 could increase migration and invasion of thyroid cancer and was related to the expression of CD44, ABCG2, snail and vimentin. Therefore, we suppose that up-regulated VCAM1 decreases treatment sensitivity of BRAF inhibition by inducing cancer stem-like properties of thyroid cancer, thus targeting VCAM1 or cancer stem-like properties may improve the efficiency of BRAF inhibition. This project is designed to investigate biological function of VCAM1, as well as its clinicopathological and prognostic value in thyroid cancer. We will also investigate how VCAM1 regulates stem-like properties of thyroid cancer and decreases the sensitivity of BRAF inhibition. Furthermore, we aim to increase BRAF inhibition efficiency by regulating VCAM1 expression and stem-like properties of thyroid cancer. The best combined therapy will be tested in cancer cell lines and verified in xenograft models. This project firstly proposes the crucial role of VCAM1 during BRAF inhibition in thyroid cancer, and will provide evidences to improve efficiency of BRAF inhibition in clinical treatment of thyroid cancer.

难治性甲状腺癌因缺少有效治疗手段而成为甲状腺癌的主要死因。BRAF突变是甲状腺癌最重要的分子事件之一，BRAF抑制剂在恶性黑色素瘤中疗效显著，但在甲状腺癌中反应率低且机制未明。VCAM1是一种与肿瘤转移、血管生成及化疗敏感性密切相关的跨膜糖蛋白粘附分子，VCAM1是否影响靶向BRAF敏感性尚不明确。课题组前期发现靶向BRAF诱导上调的VCAM1明显降低甲状腺癌靶向BRAF敏感性、增加肿瘤运动侵袭能力，并与干细胞标志物和上皮间质转化蛋白表达相关。由此推测：诱导上调的VCAM1通过调控干细胞特性降低甲状腺癌靶向BRAF敏感性，抑制VCAM1和干细胞特性可提高靶向BRAF疗效。本课题旨在评估VCAM1在甲状腺癌临床病理及预后中的价值；初步探索VCAM1表达及其降低甲状腺癌靶向BRAF敏感性的机制；筛选提高靶向BRAF疗效的联合方案并在裸鼠荷瘤模型中验证，这将为提高靶向BRAF的疗效提供重要依据。

难治性甲状腺癌因缺少有效的治疗手段而成为甲状腺癌患者的主要死因。BRAF突变是甲状腺癌最重要的分子事件之一，但BRAF抑制剂在甲状腺癌中反应率低且机制未明。VCAM1是一种与肿瘤转移、血管生成及化疗敏感性密切相关的跨膜糖蛋白粘附分子。本课题发现并证实VCAM1上调与甲状腺癌靶向BRAF敏感性下降密切相关，过表达VCAM1可促进甲状腺癌细胞迁移、侵袭，并与淋巴结转移密切相关。进一步研究显示VCAM1上调增强甲状腺癌细胞的成球能力、上调ALDH酶活性，增加侧群细胞比例，Western blot显示干细胞相关蛋白ALDH1A1、oct4、sox2的表达上调，因此VCAM1可诱导甲状腺癌干细胞特性。代谢相关研究显示VCAM1上调后甲状腺癌细胞葡萄糖摄取增多、乳酸和ATP产生增加，Western blot显示葡萄糖转运蛋白Glut1、Glut3以及糖酵解相关蛋白ENO1-3、HK2、PDK1、GPI、IDH2表达上调。因此VCAM1可增强甲状腺癌细胞糖酵解，而糖酵解途径激活是肿瘤干细胞代谢的重要特征。进一步研究分子机制发现，过表达VCAM1后stat3、JAK1/2、SRC、notch通路激活，而VCAM1的上调与p-Akt／Akt通路活化相关，Akt抑制剂可逆转上调的VCAM1表达。Akt抑制剂联合靶向BRAF可增加细胞凋亡、增强抗肿瘤效应。本课题明确了VCAM1对甲状腺癌生物学行为及靶向BRAF敏感性的影响，发现VCAM1表达可诱导甲状腺癌干细胞特性、增强糖酵解，提示糖酵解诱导的肿瘤干细胞特性是降低靶向BRAF敏感性的潜在机制，进一步探索分子机制发现Akt信号通路的活化与VCAM1表达密切相关，联合Akt抑制剂可增强靶向BRAF的抗肿瘤效应，该项目初步探索靶向BRAF耐药的分子机制，为难治性甲状腺癌的临床诊治提供了新的思路和依据。