Role of liver fluke granulin in cholangiocarcinogenesis

描述（由申请方提供）：食源性肝吸虫寄生虫猫尾后睾吸虫和华支睾吸虫感染是泰国、老挝和东亚其他感染流行地区胆管癌（CCA）、胆管癌的强风险因素。正如世界卫生组织的国际癌症研究机构所确定的那样，人类恶性肿瘤与真核病原体之间的联系没有比CCA和肝吸虫感染更强的联系。CCA通常出现较晚，对诊断来说是一个挑战，容易转移，死亡率高-这些特征突出了了解食源性寄生虫感染为何以及如何导致毁灭性肝癌的必要性。慢性感染O.灵猫体对CCA的促增殖作用是多因素的，但一个重要因素似乎是具有促有丝分裂特性的寄生虫蛋白分泌到胆管中，驱动胆管和肝细胞的局部增殖并产生致瘤环境。我们 已经在寄生虫的排泄/分泌（ES）产物中鉴定了人颗粒蛋白的同源物，人颗粒蛋白是参与细胞增殖和伤口愈合的有效生长因子。O.被称为Ov-GRN-1的灵猫颗粒蛋白广泛表达于吸虫组织中，包括肠和被膜。Ov-GRN-1在感染O.维韦里尼。重组Ov-GRN-1刺激小鼠成纤维细胞和人CCA细胞系在纳摩尔浓度下的增殖，该浓度可被MAPK激酶抑制剂抑制。抗Ov-GRN-1抗体可抑制O.提示Ov-GRN-1是存在于O.灵猫ES.这是第一次报告的分泌生长因子从寄生虫，诱导宿主细胞增殖。值得注意的是，这些新发现支持了这种吸虫蛋白在建立可能最终表现为CCA的致瘤环境中的作用。在这里，我们计划测试这一假设，特别强调Ov-GRN-1在细胞周期进展和伤口修复中的作用。我们还建议使用蛋白质组学和基因组微阵列方法来表征胆管细胞和CCA细胞系对Ov-GRN-1的转录和翻译反应，以了解肝吸虫颗粒蛋白驱动宿主细胞增殖的信号传导和其他途径。此外，我们建议使用基因沉默和接种仓鼠，以确定是否Ov-GRN-1是主要的，甚至是唯一的，寄生虫分泌的生长因子。这些研究将确定Ov-GRN-1是否是这种寄生虫的阿基里斯之踵，可以被用作抗吸虫和抗癌疫苗。

DESCRIPTION (provided by applicant): Infection with the food-borne liver fluke parasites Opisthorchis viverrini and Clonorchis sinensis is a strong risk factor for cholangiocarcinoma (CCA), bile duct cancer, in Thailand, Laos and other locations in East Asia where infection is endemic. As determined by the World Health Organization's International Agency for Research on Cancer, no stronger link exists between human malignancy and a eukaryotic pathogen than that of CCA and liver fluke infection. CCA usually presents late, is a challenge for diagnosis, metastasizes readily and has high mortality - features highlighting the necessity to understand why and how infection with a food- borne parasite leads to a devastating liver cancer. The transformation from chronic infection with O. viverrini to CCA is multi-factorial, but one importan factor appears to be secretion into the bile ducts of parasite proteins with mitogenic properties, driving local proliferation of biliary and hepatic cells and creating a tumorigenic environment. We have identified a homologue of human granulin, a potent growth factor involved in cell proliferation and wound healing, in the excretory/secretory (ES) products of the parasite. O. viverrini granulin, termed Ov-GRN-1, is widely expressed in fluke tissues, including gut and tegument. Furthermore, Ov-GRN-1 locates in situ to the surface of biliary epithelial cells of hamsters experimentally infected with O. viverrini. Recombinant Ov-GRN-1 stimulates proliferation of murine fibroblasts and human CCA cell lines at nanomolar concentrations that can be inhibited by MAPK kinase inhibitors. Antibodies to Ov-GRN-1 inhibited the ability of O. viverrini ES products to induce proliferation of mammalian cell lines in vitro, indicating that Ov-GRN-1 is the major growth factor present in O. viverrini ES. This was the first report of a secreted growth factor from a parasitic worm that induces proliferation of host cells. Significantly, these new findings support a role for this fluke protein in establishment of a tumorigenic environment that may ultimately manifest as CCA. Here we plan to test this hypothesis with a particular emphasis on the role of Ov-GRN-1 in cell cycle progression and wound repair. We propose also to characterize transcriptional and translational responses of cholangiocytes and CCA cell lines to Ov-GRN-1 using proteomic and genomic microarray approaches to understand signaling and other pathways by which liver fluke granulin drives host cell proliferation. In addition, we propose to use gene silencing and vaccination of hamsters to determine whether Ov-GRN-1 is the major, or even sole, growth factor secreted by the parasite. These studies will determine whether Ov-GRN-1 is the Achilles' heel of this parasite that could be exploited as an anti-fluke and indeed anti-cancer vaccine.