RO1: A novel and targeted approach to inhibit invasion and angiogenesis

描述（由申请人提供）： 在许多实体瘤中，胰腺癌（PC）具有最差的预后，并且它是美国所有癌症死亡的第四大原因，这可能部分是由于PC细胞在疾病的早期过程中协调“打开开关”以进行迁移、侵袭、血管生成和转移过程的能力。PC的发展是非常有利的，因为炎症事件的启动，如在人PC中发现的广泛促结缔组织增生基质（纤维形成）的存在所表明的，部分是由PDGF过表达、活性氧（ROS）的产生和NF-：B的活化所促成的。此外，EGFR和Notch-1信号传导的激活还导致NF-：B的激活，并进而调节NF-：B下游信号传导分子，例如考克斯-2、MMP-9、uPA和VEGF等，已知它们与迁移、侵袭、血管生成和转移有因果关系，最终导致诊断为PC的患者的存活率差。当使用无毒的饮食化学预防剂3，3 ′-二吲哚基甲烷（DIM），特别是BR-DIM（以下称为B-DIM，由BioResponse，Inc.提供的具有更高生物利用度的配制DIM）进行实验时，我们发现B-DIM是PDGF-D（一种最近鉴定的PDGF）、EGFR、Notch-1和NF-：B的有效抑制剂，其反过来抑制B-DIM处理的细胞中MMP-9、uPA、PGE 2和VEGF的分泌。基于我们的研究结果，我们假设使用B-DIM将作为一种新的方法，通过该方法可以抑制细胞迁移、侵袭和血管生成的过程。我们将通过实现以下三个具体目标来检验我们的假设。在目的-1中，我们将通过分析B-DIM在阻止细胞生长、肿瘤细胞迁移、侵袭和凋亡中对PDGF-D、EGFR、Notch-1、NF-：B、uPA、MMP-9、VEGF、考克斯-2（PGE 2）的作用，以及通过使用来自B-DIM处理的细胞的条件培养基在体外和还通过塞试验在体内测试的B-DIM对血管生成的作用，来彻底研究体外B-DIM处理PC细胞的结果。目的二：通过敲低和过表达的方法，研究PDGF-D、EGFR、Notch-1、NF-：B、uPA、MMP-9、VEGF和考克斯-2在体外PC细胞中介导B-DIM生物学活性的分子机制和特异性作用。在第三个具体目的中，我们将在SCID小鼠以及转基因小鼠模型中使用原位PC模型测试B-DIM的体内抗肿瘤活性，并测试抗肿瘤活性是否与肿瘤组织中EGFR、NF-κ B B、uPA、MMP-9、VEGF、PDGF-D和考克斯-2的下调相关。此外，我们将测试这些信号通路的失活是否会导致PC细胞对常规治疗药物的敏感性。拟议研究的完成将提供基于机制的证据，支持使用B-DIM预防PC细胞生长、迁移、侵袭和血管生成，以及诱导凋亡性细胞死亡。公共卫生相关性：我们的研究结果可以帮助设计创新的预防和/或治疗方法，以挽救被诊断患有这种致命疾病的患者的生命。该项目的重点是阐明化学预防剂（B-DIM）可以防止胰腺肿瘤侵袭和血管生成，从而抑制肿瘤进展的机制。我们假设B-DIM将下调PDGF-D信号传导等，从而使PDGF-D下游信号传导失活，从而抑制细胞生长、侵袭和血管生成，并抑制肿瘤进展。我们将使用分子方法和动物模型研究通过三个具体目标来验证我们的假设。

DESCRIPTION (provided by applicant): Among many solid tumors, pancreatic cancer (PC) has the worst prognosis and it is the fourth leading cause of all cancer deaths in the United States, which could be partly due to the ability of PC cells to orchestrate in "turning-on the switch" for migration, invasion, angiogenesis and metastatic processes during the early course of the disease. The development of PC is strongly favored due to initiation of inflammatory events as suggested by the presence of extensive desmoplastic stroma (fibrogenesis) found in human PCs, partly contributed by PDGF over-expression, generation of reactive oxygen species (ROS) and activation of NF-:B. In addition, activation of EGFR and Notch-1 signaling also leads to the activation of NF-:B and, in turn, regulates NF-:B downstream signaling molecules such as COX-2, MMP-9, uPA and VEGF, among others, that are known to be causally associated with migration, invasion, angiogenesis and metastasis, ultimately leading to poor survival of patients diagnosed with PCs. While conducting experiments using a non-toxic dietary chemopreventive agent, 3,3'-diindolylmethane (DIM), especially BR-DIM (hereafter known as B-DIM, a formulated DIM with greater bioavailability supplied by BioResponse, Inc.), we found that B-DIM is a potent inhibitor of PDGF-D (a recently identified PDGF), EGFR, Notch-1 and NF-:B which, in turn, inhibits MMP-9, uPA, PGE2 and VEGF secretion in B-DIM treated cells. Based on our results, we hypothesize that the use of B-DIM will serve as a novel approach by which the processes of cell migration, invasion and angiogenesis could be inhibited. We will test our hypothesis by accomplishing the following three specific aims. In aim-1, we will thoroughly investigate the consequence of B-DIM treatment of PC cells in vitro by analyzing the effect of B-DIM on PDGF-D, EGFR, Notch-1, NF-:B, uPA, MMP-9, VEGF, COX-2 (PGE2) in preventing cell growth, tumor cell migration, invasion and apoptosis, and the effect of B-DIM on angiogenesis tested by using conditioned media from B-DIM treated cells in vitro and also by plug assay in vivo. In aim-2, we will investigate the molecular mechanism(s) and the specific contribution of PDGF-D, EGFR, Notch-1, NF-:B, uPA, MMP-9, VEGF and COX-2 in mediating the biological activity of B-DIM in PC cells in vitro by applying knock-down and over-expression approaches. In the third specific aim, we will test the in vivo anti-tumor activity of B-DIM using an orthotopic PC model in SCID mice as well as in transgenic mouse models, and test whether the anti- tumor activity could be correlated with the down-regulation of EGFR, NF-:B, uPA, MMP-9, VEGF, PDGF-D and COX-2 in tumor tissues. Moreover, we will test whether the inactivation of these signaling pathways could lead to sensitization of PC cells to conventional therapeutic agents. The completion of the proposed study will provide mechanism-based evidence in support of the use of B-DIM for preventing PC cell growth, migration, invasion and angiogenesis, and induction of apoptotic cell death. PUBLIC HEALTH RELEVANCE: Our results could aid in designing innovative preventive and/or therapeutic approaches for saving lives of patients diagnosed with this deadly disease. This project is focused on elucidating the mechanism by which a chemopreventive agent (B-DIM) could prevent pancreatic tumor invasion and angiogenesis, resulting in the inhibition of tumor progression. We hypothesize that B-DIM will down regulate PDGF-D signaling among others, which in turn will inactivate PDGF-D downstream signaling, resulting in the inhibition of cell growth, invasion and angiogenesis, and the inhibition of tumor progression. We will test our hypothesis by three specific aims using molecular approaches and animal model studies.