Mechanisms of the Stromal Response to Smoothened Inhibition in Pancreatic Cancer

描述(申请人提供)：Hedgehog信号通路在多种癌症中过度表达，导致HH反应细胞中的靶基因过度激活。1在许多癌症中，反应细胞是间质成纤维细胞，位于HH配体表达的上皮细胞附近。2-5这种旁分泌机制是上皮细胞和间充质细胞之间发生的许多细胞间相互作用之一。通过它作为其他转录因子的主要调节因子的作用，HH途径的激活可以对细胞的增殖、行为和生存产生极其强大的影响。在胰腺导管腺癌(PDA)中，从肿瘤上皮细胞到成纤维细胞的旁分泌信号控制着间质结缔组织增生的发展和维持，这是这些肿瘤的一个非常显著的特征。4-7我们先前发现，PDA中的间质结缔组织增生通过干扰药物的输送而导致原发的化疗耐药。7用靶向平滑抑制物(Smo)抑制HH途径可以耗尽基因工程小鼠模型中胰腺肿瘤的间质。这起到了促进药物输送到肿瘤实质的作用，最终帮助了他们的治疗并延长了生存时间。然而，Smo抑制剂靶向促结缔组织发育的确切分子机制尚不清楚。为了更多地了解Smo抑制剂如何影响基质细胞，我们将在PDA的基因工程小鼠模型中研究Smo抑制的功能和分子后果。我们将在胰腺肿瘤组织中对下游转录因子Gli2进行CHIP-SEQ，并将结果与Smo抑制诱导的全局基因表达的变化进行比较，从而深入了解启动子与Gli转录因子结合的差异调控基因。抑制Smo在胰腺肿瘤中的另一个作用是恢复组织血管形成，这在胰腺癌中是奇怪的低。这一矛盾的结果与已知的HH途径在血管生成中的作用是不一致的。我们假设，由于成纤维细胞提供的抗血管生成信号的释放，血管生成发生在Smo抑制之后。我们将通过使用不依赖刺猬的耗尽基质的方法来测试这一点，并使用来自基因工程小鼠的组织来检查几种可能的基质介导的机制。这些实验将利用我们在小鼠建模、临床前治疗和分子生物学方面的专业知识来确定在胰腺癌和其他使用旁分泌HH信号的系统中抑制Smo的基本机制。目前，已经启动了25项不同的临床试验，以研究Smo抑制剂在包括PDA在内的9种不同癌症中的作用。这里提出的实验将有助于我们理解他们的结果，并有助于未来试验的设计。更广泛地说，我们将学习上皮细胞和间充质细胞之间的关系，以及在癌症中促进间质促结缔组织发育的途径。

DESCRIPTION (provided by applicant): The Hedgehog signaling pathway is overexpressed in a variety of cancers, leading to hyperactivation of target genes in Hh responding cells.1 In a number of cancers, the responding cells are stromal fibroblasts that are found near Hh ligand-expressing epithelial cells.2-5 This paracrine mechanism is one of many cell-cell interactions that take place between epithelial and mesenchymal cells. Through its role as a master regulator of other transcription factors, Hh pathway activation can have extremely potent effects on cell proliferation, behavior and survival. In pancreatic ductal adenocarcinoma (PDA), paracrine signaling from neoplastic epithelial cells to fibroblasts controls the development and maintenance of stromal desmoplasia, a very prominent feature of these tumors.4-7 We previously found that stromal desmoplasia in PDA contributes to primary chemoresistance by interfering with drug delivery.7 Inhibiting the Hh pathway with a targeted inhibitor of Smoothened (Smo) depleted the stroma from pancreatic tumors arising in a genetically engineered mouse model. This had the effect of facilitating drug delivery to the tumor parenchyma, ultimately aiding in their treatment and leading to prolonged survival. However, the precise molecular mechanisms by which Smo inhibitors target desmoplasia is unclear. To learn more about how Smo inhibitors affect stromal cells, we will examine the functional and molecular consequence of Smo inhibition in a genetically engineered mouse model of PDA. We will perform ChIP-SEQ for the downstream transcription factor Gli2 on pancreatic tumor tissues and compare the results to changes in global gene expression induced by Smo inhibition, providing insight into the differentially regulated genes whose promoters are bound by Gli transcription factors. Another effect of Smo inhibition in pancreatic tumors is the restoration of tissue vascularization, which is oddly low in pancreatic cancer. This paradoxical result is at odds with the known roles of the Hh pathway during angiogenesis. We hypothesize that angiogenesis occurs after Smo inhibition due to the relief of an anti- angiogenic signal provided by fibroblasts. We will test this by using hedgehog-independent means of depleting the stroma, and by examining several possible stroma-mediated mechanisms using tissues derived from genetically engineered mice. These experiments will leverage our expertise in mouse modeling, preclinical therapeutics and molecular biology to determine the basic mechanisms of Smo inhibition in pancreatic cancer and other systems employing paracrine Hh signaling. Currently, 25 different clinical trials have been initiated to investigate Smo inhibitors in 9 different cancers, including PDA. The experiments proposed here will aid our understanding of their results and assist in the design of future trials. More generally, we will learn about the relationship between epithelial and mesenchymal cells and the pathways that promote stromal desmoplasia in cancer.