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

说明（由申请人提供）：吲哚-3-甲醇（I3 C），一种从芸苔属蔬菜如花椰菜、卷心菜和布鲁塞尔芽中获得的吲哚甲醇化合物，在广泛的人类癌症中表现出有效的抗增殖特性，具有可忽略的毒性或副作用水平。我们已经证明，I3 C及其更有效和更稳定的衍生物1-苄基-I3 C，触发互补的转录，细胞信号传导和酶级联反应，控制癌细胞生长，凋亡，细胞迁移和体内肿瘤生长的人癌细胞。我们的研究最初确立了丝氨酸蛋白酶弹性蛋白酶作为人类生殖癌细胞中第一个已知的I3 C靶蛋白，这为确定其他癌细胞类型中吲哚甲醇抗增殖信号传导的机制提供了重要的实验基础。在人黑色素瘤细胞中，我们观察到I3 C诱导G1期细胞周期停滞，并通过稳定PTEN肿瘤抑制蛋白水平来抑制细胞存活信号传导，然后通过其双重脂质/蛋白磷酸酶活性破坏Akt信号传导网络，包括NFkB转录活性的失活和NFkB靶基因如细胞周期蛋白D1的减弱表达。I3 C通过抑制NEDD 4 -1介导的PTEN泛素化来触发这种反应，并且使用晶体结构NEDD 4 -1的计算机模拟3-D模拟预测I3 C特异性与NEDD 4 -1的HECT结构域相互作用，该结构域是负责E3泛素连接酶活性的结构域。该提议将检验以下假设：NEDD 4 -1 E3泛素连接酶，其选择性地泛素化和信号转导PTEN抑制蛋白的26 S蛋白酶体降解，被I3 C直接抑制，并且代表介导人黑素瘤细胞的I3 C抗增殖反应的新的吲哚甲醇靶蛋白。 该提议的一个目的是利用体外泛素化、I3 C结合和蛋白质（或结构域）相互作用测定来确定I3 C抑制NEDD 4 -1 E3泛素连接酶活性的机制。诱变策略将改变计算机模拟定义的I3 C相互作用位点，以定义I3 C结合的精确氨基酸需求，并产生新的I3 C耐药形式的NEDD 4 -1。第二个目标将在功能上表征I3 C激活的抗增殖和促凋亡级联反应，这是由于在具有不同表型的人黑色素瘤细胞中PTEN蛋白的稳定性。此外，基于I3 C与NEDD 4 -1的相互作用以及其稳定PTEN蛋白和破坏Akt/NFkB信号传导的能力，将鉴定出更有效和选择性的I3 C衍生物。第三个目的将表征吲哚甲醇化合物和阿司匹林的组合在细胞和体内环境中在黑素瘤细胞中的抗癌作用。我们提出的研究将建立临床前基础，这是需要最终开发新的和低成本的基于I3 C的人类皮肤癌的治疗策略。

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.