Role of liver fluke granulin in cholangiocarcinogenesis

描述（由申请人提供）：在泰国、老挝和东亚其他感染流行的地区，食源性肝吸虫寄生虫 Opisthorchis viverrini 和支睾吸虫的感染是胆管癌 (CCA)、胆管癌的强烈危险因素。世界卫生组织国际癌症研究机构确定，人类恶性肿瘤与真核病原体之间的联系最密切的莫过于 CCA 和肝吸虫感染。 CCA 通常发病较晚，对诊断来说是一个挑战，容易转移且死亡率高，这些特点凸显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。 从 O. viverrini 慢性感染到 CCA 的转变是多因素的，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创造致瘤环境。我们 已经在寄生虫的排泄/分泌（ES）产物中鉴定出了人颗粒蛋白的同源物，颗粒蛋白是一种参与细胞增殖和伤口愈合的有效生长因子。 O. viverrini 颗粒蛋白，称为 Ov-GRN-1，广泛表达于吸虫组织中，包括肠道和外皮。此外，Ov-GRN-1 原位定位于实验感染 O. viverrini 的仓鼠的胆管上皮细胞表面。重组 Ov-GRN-1 在纳摩尔浓度下刺激小鼠成纤维细胞和人 CCA 细胞系的增殖，该浓度可被 MAPK 激酶抑制剂抑制。 Ov-GRN-1 抗体抑制 O. viverrini ES 产品在体外诱导哺乳动物细胞系增殖的能力，表明 Ov-GRN-1 是 O. viverrini ES 中存在的主要生长因子。这是首次报道寄生虫分泌的生长因子可诱导宿主细胞增殖。值得注意的是，这些新发现支持了这种吸虫蛋白在建立致瘤环境中的作用，最终可能表现为 CCA。 在这里，我们计划检验这一假设，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，我们建议利用基因沉默和仓鼠疫苗接种来确定 Ov-GRN-1 是否是寄生虫分泌的主要甚至唯一的生长因子。这些研究将确定 Ov-GRN-1 是否是这种寄生虫的致命弱点，可以用作抗吸虫疫苗，甚至抗癌疫苗。 公共卫生相关性：长期感染肝吸虫（一种食源性寄生虫）会导致胆管癌 (CCA)，这是一种预后不良的肝癌。我们已经从这些寄生虫中鉴定出一种可能导致这种癌症的蛋白质。在这里，我们建议研究这种寄生虫蛋白（称为颗粒蛋白）在癌症中的作用，这可能会导致吸虫感染和 CCA 的新治疗和控制。

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. PUBLIC HEALTH RELEVANCE: Long term infection with liver fluke - a food-borne parasitic worm - leads to cholangiocarcinoma (CCA), a form of liver cancer with a dismal prognosis. We have identified a protein from these parasites that may cause this cancer. Here we propose to investigate the role of this parasite protein (termed granulin) in cancer, which may lead to new treatments and control for fluke infection and CCA.