Investigation of an anti-cancer phytochemical targeting Nrf2

描述（由申请人提供）：转录因子Nrf2通过上调抗氧化反应元件（ARE）基因编码抗氧化酶、解毒酶、外源转运蛋白和应激反应蛋白，成为细胞保护机制的主要调节因子。Keap1是基于Cullin3 （Cul3）的E3泛素连接酶的底物衔接蛋白，密切调节Nrf2-ARE信号通路。在基础条件下，Nrf2不断成为keap1介导的泛素化和随后的蛋白酶体降解的靶标，从而在所有人体器官中维持较低的组成水平。当该通路被激活时，Keap1-Cul3-Rbx1 E3连接酶的酶活性被抑制，导致Nrf2的稳定和Nrf2下游基因的激活。自1999年发现Nrf2- keap1 - are信号通路以来，Nrf2一直被视为保护我们免受氧化应激相关疾病（包括癌症）的“良好”转录因子。Nrf2的化学预防特性已经通过以下两个事实得到了很好的证明：(i)许多被充分研究的化学预防化合物通过激活Nrf2- are信号通路来激发它们的活性；（ii） Nrf2缺失的小鼠对化学致癌物高度敏感，不再受化学预防化合物的保护。矛盾的是，Nrf2的“阴暗面”最近被揭示了出来。例如，已经在几种类型的肿瘤和癌细胞系，特别是非小细胞肺癌（NSCLC）中发现了破坏keap1介导的Nrf2负调控的体细胞突变，导致Nrf2的高组成水平。此外，越来越多的证据表明，Nrf2有助于化疗耐药，这是癌症治疗的主要障碍。Nrf2“阴暗面”的发现清楚地表明，迫切需要识别Nrf2抑制剂，并将其开发成可药物化合物，以提高癌症治疗的疗效。我们使用实验室建立的稳定细胞系MDA-MB-231-ARE-Luc筛选了大量抑制ARE-荧光素酶活性的天然产物，该细胞系含有依赖于ARE（来自GST-Ya）的荧光素酶基因。利用这种方法，我们已经确定了一种能够抑制ARE-luciferase活性的植物提取物。此外，一种纯化合物brusatol已经从提取物中分离出来，并被发现可以抑制Nrf2的蛋白水平，并表现出有效的抗癌活性。到目前为止，我们已经获得了大量的初步数据，表明brusatol在体外使几种癌细胞系对化疗药物增敏，更重要的是，brusatol在体内以nrf2依赖的方式使肺癌异种移植物对顺铂增敏。基于brusatol特异性抑制Nrf2并使培养的癌细胞和异种移植物对顺铂治疗增敏的能力，我们假设brusatol可以通过抑制Nrf2依赖的保护机制使癌细胞对化疗药物增敏，从而增强当前癌症治疗的疗效。本研究的目的是利用临床前肺癌模型进一步表征brusatol的抗癌特性，并描述brusatol的分子靶点和机制作用。该研究不仅为Nrf2抑制剂发展成为抗化疗耐药的治疗药物提供了框架，而且为该领域的基础研究提供了第一个Nrf2抑制剂，这两者都将对全球人类健康产生深远的影响。因此，将追求以下三个目标：目标#1。brusatal介导的Nrf2抑制的机制作用表征Nrf2主要受Keap1-Cul3-Rbx1 E3连接酶在蛋白水平上通过泛素化和降解进行调控。因此，我们将研究brusatol对可能增强E3连接酶活性的蛋白质的影响，例如E3连接酶的蛋白质亚基，以及控制连接酶复合物动态组装/拆卸的调节蛋白。目标# 2。确定brusatol的分子靶点对brusatol的靶蛋白进行鉴定和验证。这些蛋白的生物学功能，特别是它们与Nrf2信号通路的串扰，将被研究。最有可能的是，这些蛋白会直接或间接调节Keap1-Cul3-Rbx1 E3连接酶。目标# 3。利用LSL-KrasG12D/+小鼠癌症模型评估brusatol作为佐剂加强当前癌症治疗和对抗内在和获得性耐药的可行性将在这个概括人类肺癌发生和进展的临床前模型中进行测试。！ ”!

DESCRIPTION (provided by applicant): The transcription factor Nrf2 has emerged as a master regulator of a cellular protective mechanism by upregulating antioxidant response element (ARE)-bearing genes encoding antioxidant enzymes, detoxifying enzymes, xenobiotic transporters, and stress response proteins. Keap1, a substrate adaptor protein for a Cullin3 (Cul3)-based E3 ubiquitin ligase, tightly regulates the Nrf2-ARE signaling pathway. Under basal conditions, Nrf2 is constantly targeted for Keap1-mediated ubiquitination and subsequent proteasomal degradation to maintain a low constitutive level in al human organs. Upon activation of the pathway, the enzymatic activity of the Keap1-Cul3-Rbx1 E3 ligase is inhibited, resulting in stabilization of Nrf2 and activation of Nrf2 downstream genes. Since the discovery of the Nrf2-Keap1-ARE signaling pathway in 1999, Nrf2 has been viewed as a "good" transcription factor that protects us from oxidative stress-related diseases, including cancer. The chemopreventive property of Nrf2 has been well documented by the following two facts: (i) many of the well- studied chemopreventive compounds elicit their activities through activation of the Nrf2-ARE signaling pathway, and (ii) Nrf2-null mice are highly susceptible to chemical carcinogens and are no longer protected by chemopreventive compounds. Paradoxically, the "dark side" of Nrf2 has recently been revealed. For instance, somatic mutations that disrupt the Keap1-mediated negative regulation of Nrf2, resulting in a high constitutive level of Nrf2, have been identified in several types of tumors and cancer cel lines, especially non-small cell lung carcinoma (NSCLC). Furthermore, mounting evidence has emerged, indicating that Nrf2 contributes to chemoresistance, the major obstacle in cancer treatment. The discovery of the "dark side" of Nrf2 has clearly illustrated the urgent need to identify Nrf2 inhibitors and develop them into druggable compounds to enhance the efficacy of cancer treatments. We have screened a large number of natural products for their inhibition of ARE-luciferase activity using a stable cell line established in our lab, MDA-MB-231-ARE-Luc, containing an ARE (from GST-Ya)-dependent luciferase gene. Using this method, we have identified a plant extract that is able to inhibit ARE-luciferase activity. Furthermore, a pure compound, brusatol, has been isolated from the extract and has been found to inhibit the protein level of Nrf2 and exhibit potent anti-cancer activities. So far, we have obtained a substantial amount of preliminary data demonstrating that brusatol sensitizes several cancer cell lines to chemotherapeutic drugs in vitro and, more significantly, brusatol sensitizes lung cancer xenografts to cisplatin in vivo in an Nrf2-dependent manner. Based on the ability of brusatol to specifically inhibit Nrf2 and sensitize cultured cancer cells and xenografts to cisplatin treatments, we hypothesize that brusatol can enhance the efficacy of current cancer treatments by sensitizing cancer cells to chemotherapeutic drugs through inhibition of the Nrf2-dependant protective mechanism. The goal of the proposed research is to further characterize the anti-cancer properties of brusatol using a preclinical lung cancer model and delineate the molecular targets and mechanistic actions of brusatol. The proposed study will not only provide a framework for the development of this Nrf2 inhibitor into a therapeutic drug to combat chemoresistance, but also provide the first Nrf2 inhibitor for basic research in the field, both of which will have profound impacts on human health worldwide. Therefore, the following three aims will be pursued: Aim #1. Characterize the mechanistic actions of brusatol-mediated Nrf2 inhibition Nrf2 is primarily regulated by the Keap1-Cul3-Rbx1 E3 ligase at the protein level through ubiquitination and degradation. Therefore, we will investigate the effects of brusatol on the proteins that may enhance the activity of the E3 ligase, such as the protein subunits of the E3 ligase, as well as regulatory proteins that control the dynamic assembly/disassembly of the ligase complex. Aim #2. Determine the molecular targets of brusatol The target proteins of brusatol will be identified and verified. The biological functions of these proteins, in particular their crosstalk with the Nrf2 signaling pathway, wil be investigated. Most likely, these proteins will directly or indirectly regulate the Keap1-Cul3-Rbx1 E3 ligase. Aim #3. Evaluate the in vivo efficacy of brusatol using an LSL-KrasG12D/+ mouselung cancer model The feasibility of using brusatol as an adjuvant to enhance current cancer treatments and to combat both intrinsic and acquired resistance will be tested in this preclinical model that recapitulates the development and progression of human lung cancer. ! "!