KIT信号分子在结直肠黏液性腺癌浸润与转移中的作用与机制研究

The mechanisms of the invasion and metastasis of malignant tumors remain incompletely understood. Seeking for principal molecules regulating cell migration during embryonic development is a newly-developed insight into the invasion and metastasis of cancers. It is well known that KIT (encoded by proto-oncogene c-kit) and ETV1 deficits or loss-of-function hinder the outmigration of melanocytes and primordial germ cells. We recently demonstrated that in murine model, colorectal mucinous adenocarcinoma (CRMAC) that highly expressed KIT and ETV4 (a homology of ETV1) often extended beyond the base membrane of mucosa and invaded into submucosa. However, the role and mechanism of KIT and ETV4 in the invasion and metastasis of CRMAC is still not fully clarified. Therefore, in this study, we plan to establish AOM+DSS induced CRMAC murine model in wild type mice, KIT loss-of-function mice and ETV4 gene conditional knock-out mice in vivo, as well as collect clinic specimens from colorectal carcinoma resection, so as to identify the expressions of KIT and ETV4 in CRMAC. Moreover, the role and mechanism of KIT-ETV4-MMPs/EMT axis in the invasion and metastasis of CRMAC will be demonstrated in vitro by using corresponding inhibitors, RNAi techniques and lenti-virus mediated gene over-expression methods in colorectal cancer cell lines. Our results will provide an insight into looking for effective therapeutic targets in the treatment of CRMAC in clinic.

恶性肿瘤浸润和转移的发生机制仍不完全清楚，探究胚胎发育过程中细胞迁移的重要调控分子可为解析癌症的浸润和转移提供重要线索。已知原癌基因c-kit编码的KIT受体和ETV1分子功能障碍或丢失，可致胚胎发育中黑素细胞和原始生殖细胞的迁移受阻。我们最近的动物实验发现，结直肠黏液性腺癌高表达KIT和ETV4，且常见癌细胞浸润基膜、转移至黏膜下层等病理变化，但二者在该类肿瘤中的作用机制尚不明了。因此，本项目拟利用AOM和DSS制作野生型小鼠、KIT功能缺失小鼠、ETV4基因条件敲除小鼠的结直肠癌模型，结合临床结直肠癌患者手术标本，首先明确KIT和ETV4在结直肠癌的表达，继之，利用结直肠癌细胞系，结合多种抑制剂、RNAi基因敲降技术和慢病毒介导的基因过表达等手段，明确KIT-ETV4-MMPs/EMT信号分子轴在结直肠黏液性腺癌浸润和转移中的作用与机制，为临床寻找新的有效治疗靶点提供重要的实验依据。

结直肠黏液性腺癌（Mucinous colorectal adenocarcinoma, MCA）是临床常见的结直肠癌亚型之一，主要病理特征是肿瘤组织中含有大量的细胞外黏液，与普通结直肠腺癌相比，MCA恶性度高，易发生转移，预后差。MCA的主要病理特征为肿瘤组织中含有大量黏液，其成分和性质与杯状细胞分泌的黏蛋白MUC2相似。因此，本研究以调控杯状细胞分化及合成与分泌MUC2的分子机制为切入点，深入探讨MCA的可能发生发展机制。.我们利用c-kit功能缺失小鼠（Wads-/-）和野生型小鼠（WT）制作MCA动物模型，并收集临床肿瘤标本，检测KIT-AKT- ATOH1信号分子轴在MCA和non-MCA的表达差异与作用，初步结果表明：.1、SCF/KIT信号高度活化可致肠黏膜上皮过度增生和肿瘤，特别是MCA的发生发展，其恶性度较non-MCA高。.2、已知正常杯状细胞的分化和MUC2的合成受转录因子ATOH1调控，本研究结果显示MCA小鼠肿瘤组织KIT及其下游AKT活性明显增加，同时ATOH1含量亦明显增多，表明ATOH1具有促进MCA发生发展的作用。.3、临床MCA患者肿瘤组织KIT-AKT- ATOH1的表达和活性改变与动物模型所获结果相同。.MCA组织中细胞外黏液的聚集除与MUC2的大量产生有关外，还与肿瘤细胞分泌黏液功能增强有关。因此，我们对肿瘤细胞分泌黏液的调控机制亦进行了探讨，结果表明：.4、SCF/KIT信号及其下游分子活化具有促进杯状细胞和MCA细胞分泌黏液的作用。.5、SCF/KIT信号通过激活囊泡运输MARCKS促进杯状细胞和肿瘤细胞分泌黏液，形成MCA。.本研究从杯状细胞的分化和MUC2合成与分泌的调控分子机制入手，明确了KIT-Akt-ATOH1-MUC2信号轴在MCA发生发展中的作用与机制，即转录因子ATOH1不仅具有诱导肿瘤细胞分化为MCA细胞，同时亦具有促进肿瘤细胞合成与分泌MUC2的作用，本研究较系统地证明了MCA发生发展的分子机制，为拓展或研发针对该关键分子的靶向药物临床治疗黏液腺癌提供重要的理论和实验依据，具有潜在的应用价值。