Normal Cellular Prion Protein in Pancreatic Cancer

描述（由申请人提供）：正常细胞朊病毒蛋白（PrPC）是一种gpi锚定的膜蛋白，存在于许多细胞类型中。虽然PrPC的表达对于一组致命的神经退行性疾病（称为朊病毒疾病）至关重要，但PrPC的正常功能仍然是一个谜。我们在一组人类肿瘤细胞系中筛选了PrPC的表达，发现人类胰腺肿瘤细胞系持续表达高水平的PrPC，并在其培养上清中产生大量的PrPC。这些发现促使我们通过抗PrPC单克隆抗体（mab）免疫组化染色来研究PrPC是否在人胰导管腺癌（Pan-DAC）组织中上调。来自慢性胰腺炎和癌前病变（如胰腺上皮内瘤变（panin - 1,2,3））患者的组织被纳入对照。在正常导管细胞、慢性胰腺炎、PanIN-1或PanIN-2组织中未检测到PrPC。只有4例（14%）PanIN-3表达低水平的PrPC。在41%的泛adc中，PrPC的表达明显上调。此外，PrPC表达与预后不良相关。癌组织中表达PrPC的患者的中位生存时间为360天，远短于不表达PrPC的患者的中位生存时间（P<0.001）。此外，siRNA下调Pan-DAC细胞系BXPC-3中PrPC的表达可降低BXPC-3细胞的增殖。这些结果表明，PrPC的表达可能在人类Pan-DAC的发生中起重要作用。这一假设得到了使用转基因小鼠系的研究的支持，该小鼠系在胰腺特异性启动子Ptfia （Ptf1acre/+;K-rasG12D/+; Tgfbr2flox/flox）的控制下，具有激活的K-ras，缺失TGF2受体2。这一转基因小鼠系开发的泛adc具有与人类泛dac相似的特征。在该转基因小鼠系中，Pan-DAC中检测到PrPC的表达，而在胰腺导管细胞、Pan-IN组织和正常胰腺导管细胞中未检测到PrPC的表达。我们假设PrPC通过其配体受体激活或功能失调的细胞凋亡级联参与泛adc亚群的致癌作用。我们提出了三个具体目标，通过体外细胞模型、转基因动物模型和额外的人体组织来进一步研究PrPC在人类Pan-DAC发生中的作用。这些研究的结果将为了解这种致命疾病的发病机制提供新的见解，也可能为这种致命疾病的治疗干预确定新的靶点。公共卫生相关性：胰腺导管腺癌（Pan DAC）是美国第四大导致死亡的癌症，每年有超过30,000人死亡。所有Pan-DAC的总中位生存期为6个月，5年生存率低于10%。目前，引起泛dac的潜在机制仍然知之甚少。我们发现，人类Pan-DAC的一个亚群（41%）有正常细胞朊病毒蛋白PrPC的表达增加，并且发现PrPC表达的增加与不良的临床结果相关（P < 0.001）。我们还发现，表达更多PrPC的人Pan-DAC细胞系具有更高的体外增殖率。此外，在Pan-DAC转基因小鼠模型中，我们发现PrPC在肿瘤中也上调，但在癌前病变中不上调。因此，我们在人类Pan-DAC中的发现在体外细胞模型和动物模型中得到了概括。我们建议通过体外细胞模型、其他患者组织和转基因小鼠模型进一步研究PrPC在Pan-DAC发展中的作用。这些研究结果将为人类Pan-DAC的发病机制提供新的见解，也可能为这种致命疾病的治疗干预提供新的靶点。

DESCRIPTION (provided by applicant): The normal cellular prion protein (PrPC) is a GPI-anchored membrane protein present on many cell types. While the expression of PrPC is critical for a group of fatal neurodegenerative conditions, known as prion diseases, the normal functions of PrPC remain an enigma. We screened for PrPC expression in a panel of human tumor cell lines, and found that human pancreatic tumor cell lines consistently express high levels of PrPC and produce high amounts of PrPC in their culture supernatants. These findings lead us to investigate whether PrPC is up regulated in human pancreatic ductal adenocarcinoma (Pan-DAC) tissues by immunohistochemical staining with anti- PrPC monoclonal antibodies (Mabs). Tissues from patients with chronic pancreatitis and pre-cancerous conditions, such as pancreatic intraepithelial neoplasia (PanIN-1, 2, 3) were included as controls. PrPC is not detected in normal ductal cells, in chronic pancreatitis, in PanIN-1 or in PanIN-2 tissues. Only four cases (14%) of PanIN-3 express low levels of PrPC. PrPC expression is uniquely up regulated in 41% of Pan-ADCs. Furthermore, PrPC expression is associated with poorer prognosis. Patients with PrPC expression in carcinoma had a much shorter median survival time of 360 days compared to >1,000 days for patients without PrPC expression (P<0.001). In addition, siRNA down regulation of PrPC expression in a Pan-DAC cell line, BXPC-3 reduces the proliferation of BxPC-3 cells. These results suggest that PrPC expression may be important in the genesis of human Pan-DAC. This hypothesis was supported by studies using a transgenic mouse line, which had an activated K-ras, with deleted TGF2 receptor 2, under the control of a pancreatic specific promoter, Ptfia, (Ptf1acre/+;K-rasG12D/+; Tgfbr2flox/flox). This transgenic mouse line develops Pan-ADC with features reminiscent of human Pan-DAC. PrPC expression was detected in Pan-DAC but not in ductal cells, Pan-IN tissues or normal pancreatic ductal cells in this transgenic mouse line. We hypothesize that PrPC is involved in carcinogenesis of a subgroup of Pan-ADCs, either through its ligand-receptor activation or dysfunctional apoptosis cascade. We proposed three specific aims, using in vitro cell models, transgenic animal models and additional human tissues to further investigate the role PrPC plays in the genesis of human Pan-DAC. Results from these studies will provide new insights into the pathogenesis and may also identify new targets for therapeutic intervention of this deadly disease. PUBLIC HEALTH RELEVANCE: Pancreatic ductal adenocarcinoma (Pan DAC) is the fourth leading cancer causing deaths in the United States with more than 30,000 deaths per year. The overall median survival for all Pan-DAC is 6 months and the 5-year survival rate is less than 10%. At present, the underlying mechanisms that cause Pan-DAC are still poorly understood. We found that a subset (41%) of human Pan-DAC has increased expression of the normal cellular prion protein, PrPC, and identified that such an increase in PrPC expression is associated with poor clinical outcome (P>0.001). We also found that human Pan-DAC cell lines that express more PrPC have a higher proliferative rate in vitro. In addition, in a transgenic mouse model of Pan-DAC, we found that PrPC is also up regulated in the tumors but not in the precancerous lesions. Therefore, our findings in human Pan-DAC are recapitulated in vitro in a cell model and in an animal model. We propose to further investigate the role PrPC plays in Pan-DAC development using in vitro cell models, additional patient tissues and a transgenic mouse model. Results from these studies will provide new insights into the pathogenesis of human Pan-DAC and may also identify new targets for therapeutic intervention of this deadly disease.