Suppressing oncogenic RNA regulons using engineered zinc finger ribonucleases

在癌症中，基因表达被广泛地重新编程。这些失调的基因包括 被称为RNA调节子的亚群，由转录后机制在 RNA水平和控制肿瘤侵袭性的关键特征。为了理解分子后果， 癌症中调节失调的RNA，这项探索性的、高风险/高回报的R21提案的目标是 开发一种锌指定向的RNA裂解剂来抑制许多上调的RNA调节子 肿瘤。我们的原型将串联锌指(TZF)结构域从Tristetraprolin(TTP)连接到 核糖核酸酶RNase4(R4)。在细胞中，嵌合的TZF-R4蛋白预计会结合并迅速降解 TTP的信使核糖核酸底物，但我们的设计将允许底物专一性被系统地修改。TTP 我们选择TZF结构域作为我们的原型，因为它已经被进化地选择为靶向RNA 含有富含AU元件(ARs)的调节子，它包括许多编码细胞调节子的mRNAs 周期、血管生成和转移。此外，TTP的表达或活性经常在 人类癌症；尤其是，乳腺肿瘤中的低TTP水平与患者预后不良有关。 这项提议旨在提供TZF-R4嵌合体可以作为引导的 癌细胞中的RNA降解系统抑制一个促肿瘤的RNA调节子并减弱相关的 肿瘤细胞表型。纯化的TZF-R4在体外表现出选择性的RNA识别和切割，以及 当瞬时转染细胞时，抑制了两个已知的TTP底物mRNAs。此外，TZF-R4 在克隆的、稳定转基因的乳腺癌细胞系中表达时，显著减缓细胞的增殖。 在这些主要初步成果的基础上，将实现两个具体目标。首先，我们将使用转录组- 广泛的方法来定义当TZF-R4稳定时靶向和不稳定的RNA调节子 TZF-R4在侵袭性乳腺癌细胞模型中的表达及对多种mRNA的抑制作用 比现有技术更有效地瞄准目标。其次，我们将评估TZF-R4对乳房的影响 癌细胞的增殖、干性、侵袭、迁移和体内肿瘤的发展。 展望了这项技术的几个未来应用，包括：(I)用于 表征RNA介导的生物途径，(Ii)开发促进摄取纯化的方法 TZF-R4进入细胞，绕过了转基因，为在体内直接给药打开了可能性 试剂，(Iii)恢复或增强TTP功能以抑制肿瘤侵袭性或炎症 信号转导，以及(Iv)通过改变TZF-R4平台的RNA靶向特异性来扩大其特异性。 扩大范围的战略包括迭代或组合修改TZF部分和 用其他的RNA结合域来“引导”嵌合蛋白，创建一个可调节的靶向蛋白家族 具有长期影响的核糖核酸酶。

Gene expression is extensively reprogrammed in cancer. These dysregulated genes include subpopulations called RNA regulons that are coordinately regulated by posttranscriptional mechanisms at the RNA level and control key features of tumor aggressiveness. To understand the molecular consequences of dysregulated RNA regulons in cancer, the goal of this exploratory, high-risk/high-reward R21 proposal is to develop a zinc finger-directed RNA-cleaving agent to suppress RNA regulons that are upregulated in many tumors. Our prototype links the tandem zinc finger (TZF) domain from tristetraprolin (TTP) to the endoribonuclease RNase4 (R4). In cells, the chimeric TZF-R4 protein is expected to bind and rapidly degrade mRNA substrates of TTP, but our design will allow substrate specificity to be systematically modified. The TTP TZF domain was chosen for our prototype because it has been evolutionarily selected to target an RNA regulon containing AU-rich elements (AREs), which includes many mRNAs that encode regulators of the cell cycle, angiogenesis, and metastasis. Furthermore, TTP expression or activity is frequently suppressed in human cancers; in particular, low TTP levels in breast tumors are associated with poor patient prognosis. This proposal is aimed at providing the “proof of concept” that a TZF-R4 chimera can function as a guided RNA degradation system in cancer cells to suppress a pro-tumorigenic RNA regulon and attenuate associated tumor cell phenotypes. Purified TZF-R4 shows selective RNA recognition and cleavage in vitro, and suppressed two known TTP substrate mRNAs when transiently transfected into cells. Furthermore, TZF-R4 dramatically slows cell proliferation when expressed in a clonal, stably-transfected breast cancer cell line. Building upon these key preliminary results, two specific aims will be pursued. First, we will use transcriptome- wide approaches to define the RNA regulon that is targeted and destabilized by TZF-R4 when stably expressed in aggressive breast cancer cell models and demonstrate that TZF-R4 suppresses multiple mRNA targets more efficiently than current technologies. Second, we will assess the impact of TZF-R4 on breast cancer cell proliferation, stemness, invasion, migration, and in vivo tumor development. Several future applications of this technology are envisioned, including: (i) discovery tools for characterizing RNA-mediated biological pathways, (ii) developing methods for promoting uptake of purified TZF-R4 into cells, bypassing transfection and opening possibilities for direct in vivo administration of this reagent, (iii) restoration or augmentation of TTP function to suppress tumor aggressiveness or inflammatory signaling, and (iv) expanding the specificity of the TZF-R4 platform by altering its RNA-targeting specificity. Strategies to broaden the scope include the iterative or combinatorial modification of the TZF moiety and substitution of other RNA-binding domains to `guide' the chimeric protein, creating a tunable family of targeted ribonucleases with long-term impact.