Targeting Smad3-Ski for Therapeutic Development in Pancreatic Cancer

描述（申请人提供）：转化生长因子β (tgf - β)信号转导通路的缺陷在胰腺癌中是众所周知的；最确定的改变是Smad4的突变，最初被称为胰腺癌4中缺失（Dpc-4），发生在大约50%的人类胰腺癌中。tgf - β结合其跨膜受体导致Smad2和Smad3磷酸化，与Smad4形成异源三聚体复合物，抑制正常胰腺上皮细胞的生长。在胰腺癌细胞中，tgf - β诱导的生长抑制可以通过Smad4的缺失或抑制Smad4依赖性生长抑制来阻止。Heider及其同事最近报道，胰腺癌细胞中Smad依赖性生长阻滞被Ski蛋白的过度表达所抑制，Ski蛋白是一种已知可直接与Smad结合以抑制信号传导的蛋白。11例人类胰腺癌标本中有9例Ski过表达，9例正常胰腺标本中无表达。在Panc-1细胞中siRNA敲低可以恢复tgf - β诱导的p21表达和生长抑制（Heider et al., Annals of Surgery 246: 61-68, 2007）。Ski的过表达也与食管鳞状细胞癌、黑色素瘤和宫颈癌的癌症表型有关。我们提出了一个试点项目来测试假设，即Ski-Smad相互作用的药理学破坏将恢复ski过表达胰腺癌细胞中的tgf - β信号传导。我们希望这些研究可能会导致一种新的治疗胰腺癌的方法。我们已经确定了小约束肽，称为肽适体，和一个小分子配体，抑制Smad3-Ski结合。在Specific Aim 1中，我们建议优化初始适配体试剂，以破坏过表达Ski的胰腺癌细胞中Smad-Ski的相互作用，并确定smad上Ski蛋白结合的“热点”。在Specific Aim 2中，我们建议通过筛选可用的化学文库，以ski结合热点为目标，发现更多的小分子配体。在Specific Aim 3中，我们的初始活性化合物和Aim 2筛选的其他药物样化学物质将优化其效力和选择性，以恢复ski过表达的人胰腺癌细胞中tgf - β生长抑制。公共卫生相关性：大多数接受当前疗法治疗的胰腺癌患者仅存活3至6个月；这种疾病的总体5年生存率<5%。我们建议临床前研究评估胰腺癌细胞的一个新的治疗靶点，Ski-Smad蛋白相互作用。我们将使用肽适体和药物样化学物质来破坏Ski-Smad，恢复胰腺癌细胞中重要的生长抑制途径，并提供原理证明，Ski-Smad是一个有效的，可药物的靶点，用于开发新的药物来帮助胰腺癌患者。

DESCRIPTION (provided by applicant): Defects in the transforming growth factor beta (TGF-beta) signal transduction pathway are well-known in pancreatic cancer; the most well-established alteration is mutation of the Smad4, originally known as deleted in pancreatic cancer 4 (Dpc-4), which occurs in approximately 50% of human pancreatic cancers examined. TGF-beta binding to its transmembrane receptors leads to phosphorylation of Smad2 and Smad3, which form heterotrimeric complexes with Smad4 to inhibit growth of normal pancreatic epithelial cells. In pancreatic cancer cells, TGF-beta-induced growth inhibition is prevented either by loss of Smad4 or by inhibiting Smad-dependent growth arrest. Heider and colleagues recently reported that Smad- dependent growth arrest in pancreatic cancer cells is inhibited by overexpression of the protein Ski, a protein known to bind directly to Smads to inhibit signaling. Nine of 11 human pancreatic cancer specimens examined overexpressed Ski, which was not expressed in 9 normal pancreatic specimens. siRNA knockdown of Ski in Panc-1 cells restored TGF-beta-induced p21 expression and growth inhibition in culture and in a xenograft model (Heider et al., Annals of Surgery 246: 61-68, 2007). Over-expression of Ski also has been implicated in the cancer phenotype of esophageal squamous cell carcinoma, melanoma and cervical carcinoma. We propose a pilot project to test the hypothesis that pharmacological disruption of the Ski-Smad interaction will restore TGF-beta signaling in Ski-overexpressing pancreatic cancer cells. We hope that these studies might lead to a novel therapeutic approach for treating some pancreatic cancers. We have identified small constrained peptides, called peptide aptamers, and one small molecule ligand that inhibits Smad3-Ski binding. In Specific Aim 1, we propose to optimize our initial aptamer reagents for disrupting Smad-Ski interactions in pancreatic cancer cells over-expressing Ski and to define the Ski protein binding "hot spots" on Smads. In Specific Aim 2, we propose to target the Ski-binding hot spots for discovery of additional small molecules ligands through screening of available chemical libraries. In Specific Aim 3, our initial active compound and additional drug-like chemicals from the Aim 2 screens will be optimized for their potency and selectivity in restoring TGF-beta growth inhibition in Ski-overexpressing human pancreatic cancer cells. PUBLIC HEALTH RELEVANCE: Most pancreatic cancer patients treated with current therapies survive for only 3 to 6 months; the overall 5- year survival rate for this disease is <5%. We propose pre-clinical studies to evaluate a new therapeutic target in pancreatic cancer cells, the Ski-Smad protein interaction. We will use peptide aptamers and drug- like chemicals to disrupt Ski-Smad, restore an important growth inhibitory pathway in the pancreatic cancer cells, and provide proof-of-principle that Ski-Smad is a valid, druggable target for new drug development to aid pancreatic cancer patients.