Role of liver fluke granulin in cholangiocarcinogenesis

描述(由申请人提供)：感染食源性肝吸虫寄生虫和华支睾吸虫是导致胆管癌(CCA)和胆道癌的重要危险因素，在泰国、老挝和其他感染流行的东亚地区。根据世界卫生组织国际癌症研究机构的研究，在人类恶性肿瘤和真核病原体之间没有比CCA和肝吸虫感染之间更强的联系。CCA通常出现较晚，对诊断是一个挑战，很容易转移，并且死亡率很高--这些特征突出了了解感染食源性寄生虫为什么以及如何导致毁灭性肝癌的必要性。从慢性感染到CCA的转变是多因素的，但一个重要的因素似乎是具有有丝分裂特性的寄生虫蛋白分泌到胆管中，推动胆管和肝细胞的局部增殖，创造一个致癌环境。我们 已经在寄生虫的排泄/分泌产物中发现了人类颗粒蛋白的同系物，这是一种参与细胞增殖和伤口愈合的有效生长因子。Viverrini颗粒蛋白被称为Ov-GRN-1，在吸虫组织中广泛表达，包括肠道和被膜。此外，Ov-GRN-1定位于实验感染金黄地鼠的胆管上皮细胞表面。重组Ov-GRN-1在纳摩尔浓度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。抗Ov-GRN-1抗体在体外能抑制Ov-GRN-1诱导哺乳动物细胞增殖的能力，表明Ov-GRN-1是Ov-GRN-1在Ov-GRN-1中存在的主要生长因子。这是首次报道一种来自寄生虫的分泌生长因子，它可以诱导宿主细胞的增殖。值得注意的是，这些新发现支持这种吸虫蛋白在建立最终可能表现为CCA的致瘤环境中的作用。在这里，我们计划通过特别强调Ov-GRN-1在细胞周期进展和伤口修复中的作用来检验这一假说。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和其他途径。此外，我们建议使用基因沉默和接种仓鼠来确定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，可以被用作抗吸虫甚至抗癌疫苗。 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)，这是一种预后不佳的肝癌。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，这可能会导致对吸虫感染和CCA的新的治疗和控制。

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.