溶瘤病毒M1肿瘤靶向生物标志物ZAP通过下调TRAILR4水平抑制肿瘤发生

Our group found that tumor-targeting of the novel oncolytic virus M1 depends on the biomarker of tumor: Zc3hav1 (ZAP) （PNAS，2014；2017）. Pre-experiments from the applicant discovered that: ZAP exhibts deficiency in a variety of malignant tumors, and the lower the expression of ZAP, the shorter the patient's survival; Cellular and animal experiments indicated that the overexpression of ZAP inhibits the malignant phenotype of tumor cells; Knockout of ZAP induces tumors occurrence in mice, and significantly shorten survival; Preliminary mechanism studies suggest that ZAP inhibits the expression of the anti-apoptotic protein TRAILR4. These evidences strongly suggest that ZAP may function as a new tumor suppressor gene. This project intends to confirm the tumor suppressive function of ZAP on more cell lines; To utilize the constructed and obtained ZAP knockout and p53 mutant or c-myc overexpression or Apc mutant genetically engineered mice to confirm the tumor suppressor function of ZAP; To use large-scale clinical patient specimens to further confirm the relationship between ZAP expression levels and tumorigenesis and survival in patients; To elucidation the detail molecular mechanism by which ZAP inhibits tumorigenesis. This project is expected to identify and elucidate a kind of new tumor suppressor gene that provides important results for tumorigenesis; More over, the project is also expected to provide biomarker for oncolytic virus M1.

本课题组发现，新型溶瘤病毒M1的肿瘤靶向性依赖于生物标志物Zc3hav1 (ZAP)（PNAS，2014；2017）。申请者的预实验发现：ZAP在多种恶性肿瘤中低表达，且ZAP表达量越低，病人生存期越短；细胞和动物实验发现，过表达ZAP抑制肿瘤恶性表型；ZAP敲除导致小鼠发生肿瘤，生存期显著缩短；初步的机制研究提示ZAP抑制抗凋亡蛋白TRAILR4的表达。这些证据强烈提示，ZAP可能是一个新的抑癌基因，这构成了本项目的科学问题。本项目拟在更多细胞上确证ZAP的肿瘤抑制作用；利用已构建和获得的ZAP敲除且p53突变或c-myc过表达或Apc突变小鼠，确证ZAP的抑癌功能；使用大规模临床患者标本进一步确证ZAP表达水平与肿瘤发生和病人生存期间的关系；深入阐明ZAP抑制肿瘤的分子机制。本项目有望发现和阐释一种新的抑癌基因，为肿瘤的发生学提供重要理论成果；同时为溶瘤病毒M1的治疗学提供生物标志物。

免疫逃逸对于肿瘤的发生来说至关重要。目前有关肿瘤和免疫之间相互作用的研究补充了癌症遗传学以外的肿瘤病理学内容。机体是一种限制病毒感染的特殊先天免疫组成部分。然而，内在免疫是否参与肿瘤病理形成尚不清楚。在此之前，我们鉴定了一种在一组临床癌症标本中经常下调表达的锌指抗病毒蛋白——ZAP。然而，ZAP是否影响肿瘤的发展尚不清楚。在本报告中我们报道了ZAP是一种真正的肿瘤抑制子。对来源于TCGA数据库的712例患者数据进行的泛caner分析和对1552例患者组织微阵列的大规模免疫组化结果显示，在肝脏、结肠和膀胱癌患者中ZAP普遍下调并与较差的生存率相关。我们在结直肠癌细胞中异位过表达ZAP后发现细胞周期阻滞最终抑制结直肠肿瘤的恶性表型。RNA免疫沉淀和RNA衰减试验证明ZAP直接且特异性结合并降解TRAILR4转录本，进而抑制TRAILR4的表达并抑制结直肠癌细胞的侵袭性。此外，我们的CRISPR小鼠模型显示，ZAP蛋白功能缺失与APC缺失在体内协同导致恶性结直肠癌发生。总的来说，我们鉴定了抗病毒因子ZAP在结直肠肿瘤发生过程中的以前从未知道的功能，并将固有免疫与肿瘤发病机制联系起来。