NITRIC OXIDE DRIVES RETINOBLASTOMA PATHWAY CHANGES IN INFLAMMATION AND CANCER

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 虽然结肠炎与结肠癌风险增加有关，但其分子机制仍未解决。我们的长期目标是确定炎症介导的致癌过程中的中心角色、途径和机制，作为确定化学预防的关键靶点的必要前提。视网膜母细胞瘤(retinoblastoma，PRB)通路是肿瘤发生的中心通路，PRB的过度磷酸化可以驱动这一过程。我们已经证明，在小鼠和人类结肠炎中，PRB被过度磷酸化和失活，导致E2F1的释放和下游靶点的激活。我们扩大了这些研究的范围，以表明一氧化氮是慢性炎症和癌症发生的关键介质，推动了pRb的过度磷酸化和途径失调。这个 这里的具体目标是： 1.确定pRb是否是一氧化氮诱导的体外表型变化中的一个分子节点。我们将在3个结肠癌(HT29、HCT15和DLD-1)和2个pRb被siRNA敲除的正常结肠癌细胞系(NCM356和NCM460)上使用5种一氧化氮暴露系统(L-精氨酸、一氧化氮供体、共培养、诱导型一氧化氮合酶表达和结构性诱导型一氧化氮合酶表达)。将评估与癌症相关的体外终点(E2F1结合、细胞周期/增殖、凋亡、转化)。 2.确定pRb上与一氧化氮失活有关的关键磷酸化位点。我们已经用野生型和突变型Rb转导了Rb阴性的结肠癌细胞。这种突变形式对被一氧化氮修饰的位点的磷酸化失活不敏感。我们将使用相同的系统将细胞暴露在一氧化氮中，并检查与特定目标1相同的端点。 3.确定体内一氧化氮诱导的pRb过度磷酸化和失活的功能后果。 为了补充体外研究，我们提出了两个额外的问题：(I)pRb是结肠炎到癌症序列中的一个分子节点吗？为了测试这一点，我们产生了具有结肠特异突变RB的小鼠，并将在它们中诱导结肠炎。(Ii)一氧化氮是否通过体内pRb的磷酸化来推动肿瘤的发生？为了测试这一点，我们将使用含有特定目标2中描述的结肠癌细胞的小鼠异种移植瘤，其中含有野生型和突变型的pRb。众所周知，pRb和一氧化氮途径参与了肿瘤的发生。这个 这个项目的创新之处在于这样一个问题：这些通路是否相互作用，增加了与慢性结肠炎相关的结肠癌发生的风险？据我们所知，这个问题还没有被探索过。数据将确定预防与慢性炎症相关的癌症的分子靶点。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Although colitis is associated with increased colon cancer risk, the molecular mechanisms remain unresolved. Our long-term goal is to identify the central players, pathways, and mechanisms involved in inflammatory-mediated carcinogenesis as a necessary prerequisite to the identification of key targets for chemoprevention. The retinoblastoma (pRb) pathway is a central pathway in carcinogenesis and pRb hyperphosphorylation can drive this process. We have shown that pRb is hyper-phosphorylated and inactivated in mouse and human colitis, leading to a release of E2F1 and activation of downstream targets. We have extended these studies to show that nitric oxide, a key mediator of chronic inflammation and carcinogenesis, drives pRb hyperphosphorylation and pathway dysregulation. The Specific Aims here are: 1. To determine whether pRb is a molecular node in nitric oxide-driven phenotypic changes in vitro. We will use 5 systems of nitric oxide exposure (L-Arginine, nitric oxide donor, co-culture, inducible iNOS expression and constitutive iNOS expression) on 3 colon cancer (HT29, HCT15, and DLD-1), and 2 normal colon cell lines (NCM356 and NCM460) with pRb knocked down by siRNA. In vitro endpoints associated with cancer (E2F1 binding, cell cycle/proliferation, apoptosis, transformation) will be evaluated. 2. To determine key phosphorylation sites on pRb that are involved in its inactivation by nitric oxide. We have transduced Rb negative colon cancer cells with wild-type Rb and mutant Rb. This mutant form is insensitive to inactivation by phosphorylation at sites identified as being modified by nitric oxide. We will expose cells to nitric oxide using the same systems and examine the same endpoints as Specific Aim 1. 3. To determine the functional consequences of nitric oxide-induced pRb hyperphosphorylation and inactivation in vivo. To complement the in vitro studies, we propose two additional questions: (i) Is pRb a molecular node in the colitis-to-cancer sequence? To test this, we have generated mice with colon specific mutant Rb and will induce colitis in them. (ii) Does nitric oxide drive tumorigenesis through pRb phosphorylation in vivo? To test this, we will use mouse xenografts containing the colon cancer cells described in Specific Aim 2 which contain the wild-type and mutant forms of pRb. It is well established that the pRb and nitric oxide pathways are involved in carcinogenesis. The innovation of this project lies in the question: Do these pathways interact to increase the risk of colon carcinogenesis associated with chronic colitis? To our knowledge, this question has not been explored. Data will identify molecular targets for the prevention of cancers associated with chronic inflammation.