Indolecarbinol target proteins and anti-cancer signaling in human melanoma cells

描述（由申请人提供）：吲哚-3-甲醇（I3C）是一种从西兰花、卷心菜和球芽甘蓝等芸苔属蔬菜中提取的吲哚-3-甲醇化合物，在多种人类癌症中表现出有效的抗增殖特性，毒性或副作用可忽略不计。我们已经证明，I3C及其更有效和稳定的衍生物1-苄基I3C，触发互补的转录、细胞信号传导和酶级联反应，控制癌细胞的生长、凋亡、细胞迁移和人类癌细胞的体内肿瘤生长。我们的研究最初确定了丝氨酸蛋白酶弹性蛋白酶是人类生殖癌细胞中已知的第一个I3C靶蛋白，这为确定吲哚卡宾醇在其他癌细胞类型中的抗增殖信号传导机制提供了重要的实验基础。在人类黑色素瘤细胞中，我们观察到I3C通过稳定PTEN肿瘤抑制蛋白水平诱导G1细胞周期阻滞并抑制细胞存活信号，通过其双脂质/蛋白磷酸酶活性破坏Akt信号网络，包括NFkB转录活性的失活和NFkB靶基因（如cyclin D1）的表达减弱。I3C通过抑制NEDD4-1介导的PTEN泛素化来触发这种反应，并且使用NEDD4-1晶体结构的三维模拟预测I3C特异性与NEDD4-1的HECT结构域相互作用，该结构域负责E3泛素连接酶活性。本研究将验证选择性泛素化PTEN抑制蛋白26S蛋白酶体降解信号通路NEDD4-1 E3泛素连接酶被I3C直接抑制的假设，是一种介导人类黑色素瘤细胞I3C抗增殖反应的新的吲哚卡宾醇靶蛋白。本提案的目的之一是利用体外泛素化，I3C结合和蛋白质（或结构域）相互作用分析来确定I3C抑制NEDD4-1 E3泛素连接酶活性的机制。诱变策略将改变计算机定义的I3C相互作用位点，以确定I3C结合所需的精确氨基酸，并产生新的耐I3C的NEDD4-1形式。第二个目标将从功能上表征I3C激活的抗增殖和促凋亡级联反应，这是由于PTEN蛋白稳定在具有不同表型的人类黑色素瘤细胞中。此外，基于其与NEDD4-1的相互作用以及稳定PTEN蛋白和破坏Akt/NFkB信号的能力，将鉴定出更有效和选择性的I3C衍生物。第三个目标将描述吲哚卡宾醇化合物和阿司匹林在细胞和体内环境下对黑色素瘤细胞的抗癌作用。我们提出的研究将为最终开发基于i3c的新型低成本人类皮肤癌治疗策略奠定临床前基础。

DESCRIPTION (provided by applicant): Indole-3-carbinol (I3C), an indolecarbinol compound obtained from Brassica vegetables, such as broccoli, cabbage and Brussels sprouts, exhibits potent anti-proliferative properties in a wide range of human cancers with negligible levels of toxicity or side effects. We have documented that I3C, and its more potent and stable derivative 1-benzyl-I3C, triggers complementary sets of transcriptional, cell signaling, and enzymatic cascades that control cancer cell growth, apoptosis, cell migration and in vivo tumor growth of human cancer cells. Our studies originally established the serine protease elastase as the first known I3C target protein in human reproductive cancer cells, which has provided a crucial experimental foundation to define the mechanisms of indolecarbinol anti-proliferative signaling in other cancer cell types. In human melanoma cells, we have observed that I3C induces a G1 cell cycle arrest and inhibits cell survival signaling by stabilizing the PTEN tumor suppressor protein levels, which through its dual lipid/protein phosphatase activity then disrupts the Akt signaling network including the inactivation of NFkB transcriptional activity and attenuated expression of NFkB target genes such as cyclin D1. I3C triggers this response by inhibiting the NEDD4-1 mediated ubiquitination of PTEN, and in silico 3-D simulations using the crystallographic structure NEDD4-1 predicts that I3C specificity interacts with the HECT domain of NEDD4-1, which is the domain responsible for the E3 ubiquitin ligase activity. This proposal will test the hypothesis that that the NEDD4-1 E3 ubiquitin ligase, which selectively ubiquitinates and signals the 26S proteasome degradation of PTEN suppressor protein, is directly inhibited by I3C and represents a new indolecarbinol target protein that mediates I3C anti-proliferative responsiveness of human melanoma cells. One aim of this proposal will utilize in vitro ubiquitination, I3C binding and protein (or domain) interaction assays to determine the mechanism by which I3C inhibits the NEDD4-1 E3 ubiquitin ligase activity. A mutagenic strategy will alter the in silico defined I3C interaction site to define the precise amino acid requirements for I3C binding and generate novel I3C-resistant forms of NEDD4-1. The second aim will functionally characterize I3C activated anti-proliferative and pro-apoptotic cascades due to PTEN protein stabilization in human melanomas cells with distinct phenotypes. Also, more potent and selective derivatives of I3C will be identified based on their interactions with NEDD4-1 and by their ability to stabilize PTEN protein and disrupt Akt/NFkB signaling. The third aim will characterize the anti-cancer effects in melanoma cells of combinations of indolecarbinol compounds and aspirin in both cellular and in vivo contexts. Our proposed studies will establish the preclinical foundation that is needed to eventually develop novel and low cost I3C-based therapeutic strategies for human skin cancers.