Role of Heparan Sulfate-Degrading Sulfatases in Pancreatic Adenocarcinomas

描述(申请人提供)：胰腺癌是人类最致命的恶性肿瘤之一，其治疗选择非常有限。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人们的极大兴趣。在这些途径中，典型的Wnt信号通路参与了干细胞/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。胰腺肿瘤标本中激活的β-连环蛋白的存在表明，30-40%的胰腺肿瘤存在Wnt信号。此外，加州大学旧金山分校的Matthias Hebrok和他的同事发现，几种胰腺癌细胞株的体外生长需要活跃的Wnt信号。我们的探索性R21项目建议研究两种新的硫酸酯酶，称为HSulf-1和HSulf-2，在胰腺癌中Wnt信号转导中的作用以及它们对这些癌细胞的生长和致瘤性的贡献。Sulf是一种胞外酶，作用于硫酸乙酰肝素蛋白多糖(HSPGs)，以去除特定的硫酸盐修饰(氨基葡萄糖-6-O-硫酸盐)。Sulf的一个已知生物活性是调节Wnt配体与HSPGs的相互作用，并增强Wnts激活其信号转导受体的能力。我们的初步研究发现，在24个胰腺癌细胞系中有23个表达SULF1或SULF2转录本，其中4/4的细胞系和3/5的胰腺癌标本中存在Sulf-1或Sulf-2蛋白。在4个品系中，有3个品系表达了SURF-1或SURF-2的显性负型(酶失活突变体)，导致Wnt信号减弱。此外，在含有非活性Sulf蛋白的情况下，共同培养同样的三个细胞系，在体外产生了Wnt信号和细胞生长的平行减少。这项拟议的研究将使用慢病毒转导的shRNA表达来沉默代表性胰腺癌细胞系中的一个或两个Sulf。我们的目标是：1)确定硫磺沉默对这些细胞的生长和Wnt信号转导的影响；2)确定硫磺沉默对细胞在裸鼠体内形成肿瘤能力的影响。这些研究的积极结果应该会激发人们对Sulf作为治疗人类胰腺癌的可能靶点(小分子或功能阻断抗体)的浓厚兴趣。

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.