Normal Cellular Prion Protein in Pancreatic Cancer

描述(申请人提供)：正常细胞蛋白(PrPC)是一种存在于多种细胞类型的GPI锚定的膜蛋白。虽然PrPC的表达在一组致命的神经退行性疾病，即PrPC疾病中是至关重要的，但PrPC的正常功能仍然是一个谜。我们筛选了一组人类肿瘤细胞系中PrPC的表达，发现人类胰腺肿瘤细胞系持续表达高水平的PrPC，并在其培养上清液中产生大量的PrPC。这些发现导致我们用抗PrPC的单抗(MAbs)免疫组织化学染色来研究PrPC在人胰腺导管腺癌组织中是否表达上调。以慢性胰腺炎和癌前病变患者的胰腺上皮内瘤变(Panin-1、2、3)为对照。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。仅4例(14%)Panin-3表达低水平的PrPC。PrPC在41%的泛ADC中表达上调。此外，PrPC的表达与预后较差有关。在癌症中表达PrPC的患者的中位生存期为360天，而不表达PrPC的患者的中位生存期为1,000天(P&lt；0.001)。此外，siRNA下调了泛DAC细胞系BXPC-3中PrPC的表达，从而抑制了BxPC-3细胞的增殖。这些结果提示PrPC的表达可能在人泛发性DAC的发生中起重要作用。这一假说得到了在胰腺特异性启动子Ptfia(Ptf1/+；K-RasG12D/+；Tgfbr2Flox/Flox)控制下，K-ras被激活、TGF2受体缺失的转基因小鼠品系的研究的支持。这个转基因小鼠系发展出了泛ADC，其特征使人想起人类的泛DAC。在该转基因小鼠系中，PrPC在PAN-DAC中表达，而在导管细胞、PAN-IN组织和正常胰腺导管细胞中未见表达。我们推测PrPC可能通过其配体受体激活或功能失调的细胞凋亡级联反应参与了一类泛ADCs的致癌过程。我们提出了三个特定的目标，利用体外细胞模型、转基因动物模型和额外的人体组织来进一步研究PrPC在人类泛发性DAC发生中的作用。这些研究的结果将为发病机制提供新的见解，并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：胰腺导管腺癌是美国导致死亡的第四大癌症，每年有超过3万人死亡。全组中位生存期为6个月，5年生存率不到10%。目前，引起泛DAC的潜在机制仍然知之甚少。我们发现，一组(41%)的人泛-DAC增加了正常细胞内PrPC蛋白的表达，并发现PrPC蛋白的这种表达增加与不良的临床结果有关(P&gt；0.001)。我们还发现，表达更多PrPC的人泛DAC细胞株在体外具有更高的增殖率。此外，在一个转基因小鼠模型中，我们还发现PrPC在肿瘤中表达上调，但在癌前病变中不表达。因此，我们在体外细胞模型和动物模型中总结了我们在人类泛型DAC中的发现。我们建议使用体外细胞模型、额外的患者组织和转基因小鼠模型进一步研究PrPC在泛DAC发育中的作用。这些研究的结果将为人类泛DAC的发病机制提供新的见解，并可能为这一致命疾病的治疗干预确定新的靶点。

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.