A novel high content screen for regulators of ciliogenesis and Hedgehog signaling

描述（由申请人提供）：刺猬通路是控制哺乳动物细胞行为的一种基本手段，用于调节各种不同的生物事件，包括组织模式，干细胞更新和细胞增殖。所有的Hedgehog信号都依赖于原癌基因Smoothened，这种突变可导致基底细胞癌（北美最常见的癌症）和成神经管细胞瘤（儿童中最常见的实体癌）。目前在临床试验中，肿瘤已经显示出对Smoothened拮抗剂的耐药性，这表明需要其他的Hedgehog通路抑制剂。我们已经发现哺乳动物的刺猬信号平滑地移动到一种叫做初级纤毛的细胞器，这种运动对于通路激活是必要的。虽然已知几乎所有哺乳动物细胞都有一个延伸到细胞外环境的初级纤毛，但人们对这种细胞器的功能知之甚少。我们假设初级纤毛作为细胞天线，通过它传导Hedgehog信号。尽管纤毛对发育和疾病都很重要，但其产生和平滑功能的分子机制尚不清楚。为了解决我们理解中的这些空白，我们寻求进行高通量筛选，以确定可以抑制纤毛形成或平滑纤毛运动的新化合物。使用高通量显微镜和高含量分析相结合，我们将可视化和定量的定位到培养细胞的纤毛。我们将对抑制纤毛Smoothened定位的新化合物进行二次分析，以揭示该化合物是否影响纤毛的生物发生、Smoothened运输和Hedgehog途径活性等参数。这项工作将产生化学工具，药理学解剖纤毛发生和平滑调节的分子机制。此外，考虑到错误调控的刺猬信号在多种人类癌症中发挥的核心作用，这些化合物可能为开发新的化疗药物提供基础。

DESCRIPTION (provided by applicant): The Hedgehog pathway is one fundamental means of controlling mammalian cell behavior and is used to regulate a wide variety of disparate biological events including tissue patterning, stem cell renewal, and cell proliferation. All Hedgehog signaling relies on the proto-oncogene Smoothened, mutations in which can cause basal cell carcinoma, the most common cancer in North America, and medulloblastoma, the most common solid cancer among children. Tumors have displayed resistance to Smoothened antagonists currently in clinical trials, indicating a need for other inhibitors of the Hedgehog pathway. We have discovered that mammalian Hedgehog signals move Smoothened to an organelle called the primary cilium, and that this movement is necessary for pathway activation. Although it is known that almost all mammalian cells possess a single primary cilium that extends into the extracellular environment, the functions of this organelle are poorly understood. We hypothesize that the primary cilium acts as a cellular antenna, through which Hedgehog signals are transduced. Despite their importance to both development and disease, the molecular mechanisms by which the cilium is generated and by which Smoothened functions remain unclear. To address these gaps in our understanding, we seek to conduct a high-throughput screen to identify novel compounds that can inhibit cilium formation or Smoothened movement to cilia. Using a combination of high-throughput microscopy and high-content analysis, we will visualize and quantitate Smoothened localization to the cilium in cultured cells. We will subject novel compounds that inhibit Smoothened localization to the cilium to secondary assays that reveal whether the compound affects cilium biogenesis, Smoothened trafficking, and Hedgehog pathway activity, among other parameters. This work will produce the chemical tools to pharamacogenetically dissect the molecular mechanisms of ciliogenesis and Smoothened regulation. Additionally, given the central role misregulated Hedgehog signaling plays in diverse human cancers, these compounds may provide the bases for the development of novel chemotherapeutic drugs. PUBLIC HEALTH RELEVANCE: Problems in cell-cell communication can underlie diseases as diverse as birth defects and cancer. One mechanism of cell-cell communication uses Hedgehog proteins, secreted signaling proteins. Defects in Hedgehog signaling can cause birth defects, such as holoprosencephaly, and several types of cancer, including medulloblastoma. Recent evidence has revealed that Hedgehog signaling relies on the primary cilium, a poorly understood organelle present on most cells. To be able to better understand how Hedgehog signaling and the primary cilium function, we propose to conduct a screen for small molecules that perturb these processes. These chemicals may provide the basis for the development of novel therapies for Hedgehog-related cancers.