Role of Heparan Sulfate-Degrading Sulfatases in Pancreatic Adenocarcinomas

描述（由申请方提供）：胰腺癌是人类最致命的恶性肿瘤之一，治疗选择非常有限。胚胎信号通路在这种癌症和其他癌症的发生和发展中的可能作用引起了极大的兴趣。在这些途径中，典型的Wnt信号传导参与干细胞/祖细胞的增殖和自我更新，并被推测在癌症干细胞中具有类似的作用。胰腺肿瘤样品中活化的β-连环蛋白的存在表明30-40%的这些肿瘤表现出Wnt信号传导。此外，加州大学旧金山分校的Matthias Hebrok及其同事发现，活跃的Wnt信号传导是几种胰腺癌细胞系体外生长所必需的。我们的探索性R21项目提出研究两种新型硫酸酯酶HSulf-1和HSulf-2在胰腺癌中Wnt信号传导中的作用及其对这些癌细胞生长和致瘤性的贡献。硫酸是胞外酶，作用于硫酸乙酰肝素蛋白聚糖（HSPG）以去除特定的硫酸化修饰（葡糖胺-6-O-硫酸化）。硫的一种已知生物活性是调节Wnt配体与HSPG的相互作用并增强Wnt激活其信号转导受体的能力。我们的初步研究发现，SULF-1或SULF-2转录本在24个胰腺癌细胞系中的23个中表达，Sulf-1或Sulf-2蛋白在4/4的这些细胞系和3/5的胰腺肿瘤样品中存在。在这些品系中的4个中的3个中，Sulf-1或Sulf-2的显性负性形式（酶失活突变体）的表达导致Wnt信号传导的减少。此外，在无活性Sulf蛋白存在下共培养相同的三种细胞系在体外产生Wnt信号传导和细胞生长的平行减少。这项研究将采用慢病毒转导的shRNA表达来沉默代表性胰腺癌细胞系中的一种或两种Sulf。我们的目标是：1）确定Sulf沉默对这些细胞体外生长和Wnt信号传导的影响;和2）确定Sulf沉默对细胞在裸鼠中形成肿瘤的能力的影响。这些研究的积极结果应该会激发人们对Sulfs作为治疗人类胰腺癌的可能靶点（小分子或功能阻断抗体）的极大兴趣。

DESCRIPTION (provided by applicant): Pancreatic adenocarcinoma is one of the most deadly malignancies in human for which there are very limited treatment options. There is great interest in the possible role of embryonic signaling pathways in the initiation and progression of this cancer and others. Among these pathways is canonical Wnt signaling, which is involved in the proliferation and self-renewal of stem/progenitor cells and has been speculated to have a similar role in cancer stem cells. The presence of activated beta-catenin in pancreatic tumor samples suggests that 30-40% of these tumors manifest Wnt signaling. Moreover, Matthias Hebrok and colleagues at UCSF have discovered that active Wnt signaling is required for the growth of several pancreatic adenocarcinoma cell lines in vitro. Our exploratory R21 project proposes to investigate the role of two novel sulfatases, called HSulf-1 and HSulf-2, in Wnt signaling within pancreatic adenocarcinomas and their contributions to the growth and tumorigenicity of these cancer cells. The Sulfs are extracellular enzymes which act on heparan sulfate proteoglycans (HSPGs) to remove a specific sulfation modification (glucosamine-6-O-sulfation). One known biological activity of the Sulfs is to modulate the interaction of Wnt ligands with HSPGs and to potentiate the ability of the Wnts to activate their signal transduction receptors. Our preliminary studies have found the expression of SULF1 or SULF2 transcripts in 23 of 24 pancreatic adenocarcinoma cell lines and the presence of Sulf-1 or Sulf-2 protein in 4/4 of these cell lines and 3/5 of pancreatic tumor samples. Expression of a dominant negative form (enzymatically-inactive mutant) of either Sulf-1 or Sulf-2 in 3 out of 4 of these lines resulted a reduction in Wnt signaling. Furthermore, co-culturing the same three cell lines in the presence of inactive Sulf protein produced a parallel reduction in Wnt signaling and cell growth in vitro. The proposed research will employ lentivirus-transduced shRNA expression to silence one or both Sulfs in representative pancreatic adenocarcinoma cell lines. Our Aims are: 1) To determine the effects of Sulf silencing on the growth and Wnt signaling of these cells in vitro; and 2) To determine the effects of Sulf silencing on the ability of the cells to form tumors in nude mice. Positive results from these studies should stimulate considerable interest in the Sulfs as possible targets (for small molecules or function-blocking antibodies) for the treatment of pancreatic adenocarcinoma in humans.