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

描述(由申请人提供)： 在许多实体肿瘤中，胰腺癌(PC)预后最差，是美国癌症死亡的第四大原因，部分原因可能是PC细胞在疾病的早期过程中协调了迁移、侵袭、血管生成和转移过程的“开关”。在PC中发现广泛的促结缔组织间质(纤维化形成)，部分是由于PDGF的过度表达、活性氧物种(ROS)的产生和NF-B的激活而导致炎症事件的发生，因此PC的发展非常受欢迎。此外，EGFR和Notch-1信号也导致NF-：B的激活，进而调节NF-：B下游信号分子，如COX-2、MMP-9、uPA和VEGF等，这些已知与迁移、侵袭、血管生成和转移有关，最终导致被诊断为PC的患者生存不良。在使用无毒的饮食化学预防药物3，3‘-二吲哚甲烷(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方DIM)进行实验时，我们发现B-DIM是PDGF-D(一种最近发现的PDGF)、EGFR、Notch-1和NF-：B的有效抑制剂，后者反过来又抑制B-DIM处理的细胞分泌MMP-9、uPA、PGE2和VEGF。根据我们的结果，我们假设B-DIM的使用将成为一种新的方法，通过它可以抑制细胞的迁移、侵袭和血管生成过程。我们将通过实现以下三个具体目标来验证我们的假设。在AIM-1中，我们将通过分析B-DIM对PDGF-D、EGFR、Notch-1、NF-：B、uPA、MMP-9、VEGF、COX-2(PGE2)在抑制细胞生长、肿瘤细胞迁移、侵袭和凋亡方面的作用，以及B-DIM对血管生成的影响，在体外和体内用B-DIM处理的细胞条件培养液检测B-DIM对PC细胞血管生成的影响。在AIM-2中，我们将通过下调和过表达的方法，在体外研究PDGF-D、EGFR、Notch-1、NF-：B、uPA、MMP9、血管内皮生长因子和COX-2在介导PC细胞B-DIM生物学活性中的分子机制(S)及其特异性贡献。在第三个特定目标中，我们将在SCID小鼠和转基因小鼠模型上使用原位PC模型测试B-DIM的体内抗肿瘤活性，并测试其抗肿瘤活性是否与肿瘤组织中EGFR、NF：B、uPA、基质金属蛋白酶-9、血管内皮生长因子、PDGF-D和COX-2的下调有关。此外，我们将测试这些信号通路的失活是否会导致PC细胞对传统治疗药物的敏感性。这项拟议研究的完成将为使用B-DIM预防PC细胞的生长、迁移、侵袭和血管生成以及诱导凋亡细胞死亡提供基于机制的证据。公共卫生相关性：我们的结果可能有助于设计创新的预防和/或治疗方法，以挽救被诊断为这种致命疾病的患者的生命。本课题致力于阐明化学预防性药物(B-DIM)阻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，进而使PDGF-D下游信号失活，从而抑制细胞生长、侵袭和血管生成，从而抑制肿瘤进展。我们将使用分子方法和动物模型研究，通过三个特定的目标来检验我们的假设。

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.