神经节苷脂GD3合成酶为潜在肿瘤治疗靶标的研究

Invasion and metastasis are the most important characteristics of malignant tumor. Invasion and metastasis are also main reasons for the failure of cancer therapy in clinic. More and more evidences show that cancers are driven by cancer stem cells (CSCs), a distinct cell-type which is tumorigenic, capable of invasion and metastasis, and is resistant to chemotherapy. Ganglioside GD3 can regulate cell proliferation and differentiation and GD3 has been first described as a tumor-associated ganglioside since it is overexpressed in a variety of tumors. Recent study reveals that Ganglioside GD2 identifies breast cancer stem cells and promotes tumorigenesis and that GD3 synthase (GD3s) is highly expressed in GD2+ cells. Ganglioside GD2 is overexpressed among pediatric and adult solid tumors, including breast cancer, small cell lung cancer, neuroblastoma, glioma and melanoma. GD2 is thought as a new therapeutic target for human tumors. GD3 synthase regulates epithelial–mesenchymal transition and metastasis in breast cancer. Knockdown of GD3s completely abrogated tumor formation in vivo. Inhibition of GD3s expression can inhibit angiogenesis and the expression of vascular endothelial growth factor. Our study showed that overexpression of GD3s in cells increased proliferation, migration and invasion of cells. GD3s is only key enzyme for synthesis of GD3 and GD2. Inhibitor of GD3s can not only inhibit GD3s but also GD3 and GD2. GD3s inhibitors can inhibit the invasion and metastasis of tumor and inhibiting proliferation of tumor stem cell. Inhibitor of GD3s will have an important role in the treatment of tumors. We propose GD3s can be used as a potential cancer therapeutic target. Currently, there is no screening model for GD3s inhibitor and no inhibitor for GD3s activity. We will establish two high throughput screening models for inhibitor of GD3s. One is based on enzyme reaction, and the other is based on fluorescence polarization. The two models will be used to screen GD3s inhibitor using our chemical library. We will evaluate anti-tumor activity of lead compounds in vitro and in vivo. We will further prove that GD3s can become an important drug target of a tumor therapy through screening and evaluation of GD3s inhibitors. It is necessary and important to establish high throughput screening model for inhibitor of GD3s and discover inhibitor of GD3s for tumor treatment.

浸润和转移是恶性肿瘤最重要的特征之一。肿瘤干细胞对肿瘤的产生、发展、化疗抗性、转移及复发有重要作用。发现抑制肿瘤转移和肿瘤干细胞的新药对治疗肿瘤具有重要意义。GD3是最早发现和肿瘤相关的神经节苷脂。GD2是乳腺癌干细胞的生物标记，在多种肿瘤中高表达。GD3 合成酶（GD3 synthase ，GD3s）在GD2+ 乳腺癌干细胞表达很高。抑制GD3s表达的细胞在裸鼠中没有成瘤能力。我们研究显示GD3s在肿瘤转移中起重要作用。 GD3s是合成GD3 和GD2的唯一关键酶。GD3s的抑制剂既能抑制GD3s又能抑制GD3和GD2的生成。我们认为GD3s抑制剂既有抑制肿瘤的转移又有抑制肿瘤干细胞生长作用，并提出GD3s可以成为一个潜在肿瘤治疗的重要药物靶点；建立两个GD3s抑制剂高通量筛选模型进行样品筛选。我们还将对活性化合物进行抗肿瘤评价。GD3s作为治疗肿瘤靶标的研究对肿瘤新药发现具有重要意义。

恶性肿瘤严重危害人民的身体健康。浸润和转移是恶性肿瘤最重要的特征之一。发现肿瘤转移的药物靶点和发现抗肿瘤转移的新药对治疗肿瘤具有重要意义。在本项目中我们研究了GD3S在肿瘤中的表达、功能及作用机制；GD3S表达的表观遗传学调控机制；GD3S的同源建模及虚拟筛选、GD3S抑制剂高通量筛选模型的建立及筛选；GD3S的表达，纯化及GD3S蛋白晶体结构解析研究。研究发现GD3S在ER阴性和三阴性乳腺癌中高表达；GD3S的表达随着乳腺癌的发展增加。GD3S的表达与乳腺癌病人的预后密切相关。在乳腺癌GD3S的表达与GD3S基因的甲基化程度密切相关。我们还发现在黑色素细胞中过表达GD3S能促进细胞的增殖，迁移，侵袭和克隆形成。相反，在黑色素瘤细胞中抑制GD3S的表达则抑制增殖，迁移，侵袭和克隆形成。研究还发现GD3S促进肿瘤的肺转移和骨转移。GD3S促进上皮细胞向间皮细胞转化。我们成功构建了GD3S原核表达质粒和真核表达质粒并首次在大肠杆菌原核表达系统、昆虫-干状病毒真核系统表达大量高活性的GD3S蛋白。我们进一步利用真核表达的高活性蛋白建立GD3S抑制剂高通量筛选模型。我们还将昆虫-干状病毒真核表达系统表达出的大量高活性GD3S蛋白经分离、纯化后与磷酸胞苷唾液酸（CMP-Sialic Acid）一起孵育形成多种不同的晶体，为首次解析GD3S晶体结构和确定其活性位点打好一个基础。晶体结构解析也有助于GD3S抑制剂的设计和发现，有助于阐明转移单个唾液酸这类酶的催化机理。这一研究对认识GD3S在乳腺癌和黑色素瘤中作用及分子机制具有重要意义。确定GD3S为抗肿瘤药物靶点具有重要意义。